Analytical Techniques For Studying The Physical Properties of Lipid Emulsions (2012)

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Maria Lidia Herrera
Analytical Techniques
for Studying the Physical
Properties of Lipid
Emulsions
Maria Lidia Herrera
University of Buenos Aires
Faculty of Exact and Natural Sciences
Buenos Aires
Argentina
ISBN 978-1-4614-3255-5 e-ISBN 978-1-4614-3256-2

DOI 10.1007/978-1-4614-3256-2
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2012932599
Maria Lidia Herrera 2012

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Contents
1 Introduction ............................................................................................... 1
References ................................................................................................... 5
2 Nano and Micro Food Emulsions ............................................................ 7
2.1 Methods of Formation........................................................................ 7
2.1.1 Nanoemulsions ....................................................................... 7
2.1.2 Conventional Emulsions ........................................................ 10
2.2 Physical Chemical Properties ............................................................ 11
2.2.1 Nanoemulsions ....................................................................... 11
2.2.2 Conventional Emulsions ........................................................ 12
2.3 Structuring Food Emulsions .............................................................. 12
References ................................................................................................... 13
3 Methods for Stability Studies ................................................................... 15
3.1 Visual Observation ............................................................................. 15
3.2 Rheological Methods ......................................................................... 20
3.2.1 Small-Deformation Rheology ................................................ 21
3.2.2 Viscosity ................................................................................. 24
3.3 Ultrasound Profiling ........................................................................... 27
3.4 Electroacoustic Spectroscopy: z-Potential ......................................... 31
3.5 Measurement of Surface Concentration............................................. 33
3.6 Microscopic Analysis......................................................................... 34
3.6.1 Transmission Electron Microscopy (TEM) ........................... 34
3.6.2 CLSM ..................................................................................... 37
3.7 Nuclear Magnetic Resonance (NMR) Techniques............................. 39
3.8 Optical Methods ................................................................................. 43
3.8.1 Dynamic Light Scattering (DLS) ........................................... 43
3.8.2 Diffusing Wave Spectroscopy (DWS).................................... 46
3.8.3 Turbiscan ................................................................................ 48
References ................................................................................................... 55
Index ................................................................................................................. 61
v
Chapter 1
Introduction
Water and lipids are critical for sustaining life and health, but their poor miscibility
has posed a challenge for both nature and man. Emulsions are colloidal dispersions
that consist of two immiscible liquids, with one of the liquids being dispersed in the
other one. Oil dispersions in the form of small spherical droplets are stabilized in the
aqueous phase by proteins or surfactants giving an oil-in-water (O/W) emulsion.
The surface-active ingredient is adsorbed at the interface between oil and the aque-
ous phase to lower surface tension and prevent oil droplets from coming close
enough together to aggregate.
Emulsions are materials with a widespread range of applications, the most impor-
tant ones including cosmetics, foods, detergency, adhesives, coatings, paints, surface
treatment, road surfacing, and pharmaceutics (Thivilliers et al. 2008). Among edible
materials, a considerable number of natural and processed foods consist either partly
or wholly as emulsions, or have been in an emulsified state sometime during their
production, including milk, cream, fruit beverages, infant formula, soups, cake bat-
ters, salad dressings, mayonnaise, cream liqueurs, sauces, desserts, salad cream, ice
cream, coffee whitener, spreads, butter, and margarine (McClements 2005a).
The understanding and manipulation of bulk properties of emulsion systems are of
the utmost importance in the food industry. The physicochemical and sensory proper-
ties of a particular food emulsion depend on the type and concentration of ingredients
that it contains, as well as the method used to create it. In addition, shelf life, mouth-
feel, and flow properties, to name a few, are to a great extent determined by interac-
tions present among the systems constituents (Corredig and Alexander 2008).
Nanoemulsions can be distinguished from the conventional emulsions that are
currently more commonly used in the food industry in terms of their droplet size.
Conventional emulsions can be defined as having droplet radii greater than 100 nm,
whereas nanoemulsions have radii less than 100 nm (Mason et al. 2006). Conventional
emulsions tend to be cloudy or opaque in appearance because the dimensions of the
lipid droplets are on the same order as the wavelength of light (d l), so that light
scattering is relatively strong. On the other hand, nanoemulsions tend to be transpar-
ent or slightly turbid in appearance because the dimensions of the lipid droplets are
M.L. Herrera, Analytical Techniques for Studying the Physical Properties 1

of Lipid Emulsions, SpringerBriefs in Food, Health, and Nutrition 3,
DOI 10.1007/978-1-4614-3256-2_1, Maria Lidia Herrera 2012
2 1 Introduction
usually much smaller than the wavelength of light (d l), so that light scattering is
relatively weak. Conventional emulsions are often prone to gravitational separation
and droplet aggregation because of the relatively large size of the droplets. On the
other hand, nanoemulsions are usually highly stable to gravitational separation
because the relatively small droplet size means that Brownian motion effects domi-
nate gravitational forces. In addition, nanoemulsions tend to have better stability
against droplet aggregation than conventional emulsions because the strength of the
net attractive forces acting between droplets usually decreases with decreasing
droplet size, whereas the strength of the repulsive steric forces is less dependent on
size. The rheological properties of nanoemulsions follow similar trends to conven-
tional emulsions; that is, the viscosity increases with increasing droplet concentra-
tion and with droplet aggregation. Nevertheless, the viscosity of a nanoemulsion
may be appreciably greater than that of a conventional emulsion at the same lipid
concentration if it contains a thick or electrically charged interfacial layer that
increases dropletdroplet repulsion (McClements and Li 2010).
One of the main properties of a food emulsion is its stability, that is, the ability
to resist changes in its properties over time. The length of time that an emulsion
must remain stable depends on the nature of the food product. Some food emulsions
are formed as intermediate steps during a manufacturing process and therefore only
need to remain stable for a few seconds, minutes, or hours (e.g., cake batter, ice
cream mix, and margarine premix), whereas others must remain stable for days,
months, or even years prior to consumption (e.g., mayonnaise, salad dressings, and
cream liqueurs). On the other hand, the production of some foods involves a con-
trolled destabilization of an emulsion during the manufacturing process, for exam-
ple, margarine, butter, whipped cream, and ice cream (McClements 2005b).
Colloids and dispersions are inherently unstable products, but they can be consid-
ered stable if their destabilization velocity is sufficiently low compared with the
expected lifespan. Special precautions need to be taken to overcome emulsions nat-
ural tendency to demix or break. A major part of the physical stability of food emul-
sions is determined by the continuous phase, but also the distribution of droplet sizes
plays an important role. Among the major objectives of emulsion scientists working
in the food industry is to establish the specific factors that determine the stability of
each particular type of food emulsion as well as to elucidate general principles that
can be used to predict the behavior of food products or processes. Among physical
destabilization mechanisms, the most common phenomena affecting the homogene-
ity of conventional emulsions are particle migration (creaming, sedimentation) and
particle size variation or aggregation (coalescence, flocculation).
In general, the droplets in an emulsion have a different density than that of the
liquid that surrounds them, and so a net gravitational force acts on them. The parti-
cles float upwards or sink, depending on how large they are and how much less
dense or denser they may be than the continuous phase. Creaming is the migration
upward of the dispersed phase of an emulsion, while sedimentation is the downward
movement of droplets. Gravitational separation is usually regarded as having an
adverse effect on the quality of food emulsions. A consumer expects to see a prod-
uct that appears homogeneous, and therefore the separation of an emulsion into an
optically opaque droplet-rich layer and a less opaque droplet-depleted layer is
1 Introduction 3
Fig. 1.1 Confocal laser

scattering microscopy
(CLSM) images of emulsions
with 10 wt.% sunflower oil
(SFO) as fat phase and
0.5 wt.% sodium caseinate
(NaCas) kept at 22.5C for
24 h. The green color
corresponds to the fat phase
undesirable (McClements 2005b). Figure 1.1 shows an example of a confocal laser

scanning microscopy image of a sunflower/sodium caseinate emulsion that destabi-
lized by creaming. Emulsion was stabilized by 0.5 wt.% sodium caseinate. Sunflower
oil content was 10 wt.%. The image was taken 24 h after preparation. The emulsion
was still stable at that time. In an emulsion that mainly destabilized by creaming, the
fat phase appeared as individual droplets evenly distributed immediately after prep-
aration; then droplets remained as individual particles during destabilization.
Flocculation is a process in which individual particles of a suspension form aggre-
gates, while coalescence is caused by rupture of the film between two emulsion
drops or two foam bubbles. The driving force for coalescence is the decrease in free
energy resulting when the total surface area is decreased, as occurs after film rup-
ture. The former process is reversible, while the latter leads to bigger particles.
Figure 1.2 provides a confocal laser scanning image of a sunflower/sodium casein-
ate emulsion that destabilized by flocculation. In this case, the sodium caseinate
concentration was 5 wt.%. As may be noticed, fat droplets were aggregated, form-
ing flocs. In emulsion systems, the d4,3 parameter, the volume-weighted mean diam-
eter of initial emulsions, obtained from droplet size distribution expressed as
differential volume, is more sensitive to fat droplet aggregation (coalescence and/or
flocculation) than the Sauter mean diameter (d3,2) (Relkin and Sourdet 2005). When
this sample was analyzed for particle size distribution, immediately after prepara-
tion and after a week at 22.5C, there were no changes in d4,3, meaning that the
emulsion in the example destabilized by flocculation.
Emulsions may also destabilized by other mechanisms such as partial coales-
cence, phase inversion, and Ostwald ripening. Partial coalescence is usually described
as the destabilization process that occurs when fat crystals present within the thin
film separating two droplets pierce the film and bridge the surfaces, causing the
4 1 Introduction
Fig. 1.2 Confocal laser

scattering microscopy
(CLSM) images of emulsions
with 10 wt.% sunflower oil
(SFO) as fat phase and
5 wt.% sodium caseinate
(NaCas) kept at 22.5C for
24 h. The green color
corresponds to the fat phase
droplets to coalesce. If the crystallized fraction within the droplets is sufficient, the
intrinsic rigidity inhibits relaxation to the spherical shape driven by surface tension
after each coalescence event. Large clusters appear and grow by the accretion of any
other primary droplet or cluster until a rigid network made of partially coalesced
droplets is formed (Davies et al. 2000; van Aken 2001; Vanapalli and Coupland
2001; Vanapalli et al. 2002; Giermanska-Kahn et al. 2005; Thivilliers et al. 2006;
Golemanov et al. 2006; Giermanska et al. 2007; Thivilliers-Arvis et al. 2010).
Emulsion inversion refers to the swap of the dispersed phase and the continuous
phase of the emulsion. The phenomenon occurs under certain conditions, and the
process is often used as a route to make emulsions (Liu and Friberg 2010). Inversion
is a central process in emulsion technology, both as an integral part of the manufac-
turing technology and as an inevitable component in a large number of emulsion
applications, especially those involving evaporation, since the evaporation path, by
necessity, leads to inversion when the evaporation takes place predominantly from
the continuous phase (Corkery et al. 2010).
Ostwald ripening involves the transport by diffusion of molecules of the disperse
phase from small to large particles, due to the differences in Laplace pressure.
Laplace stated that the pressure at the concave side of a curved interface is higher
than the pressure at the convex side by an amount that is proportional to the interfa-
cial tension times the curvature; for a sphere, the curvature is given by the reciprocal
of its radius. The result is that the solubility of the dispersed phase in the continuous
phase is increased to a larger extent for a small particle than for a large one; hence,
small particles shrink (and will eventually disappear), and large particles grow
larger. The rate at which the transport will occur is about proportional to the solubil-
ity of the oil in the continuous phase. Given that the solubility of nearly all natural
References 5
TAG in most aqueous systems is negligible, the Ostwald ripening is not a common
destabilization mechanism in conventional food oil-in-water emulsions (Walstra
2003; Giermanska et al. 2007; Fredrick et al. 2010). Nanoemulsions, however, may
be unstable to Ostwald ripening and have to be specifically designed to prevent this
phenomenon from occurring, for example, by adding a highly water-insoluble com-
ponent (Sonneville-Aubrun et al. 2004; Wooster et al. 2008; Li et al. 2009).
As food products are very complex systems, food scientists often rely on the use
of analytical techniques to experimentally monitor changes in emulsion properties
over time. By using a combination of theoretical understanding and experimental
measurements, food manufacturers are able to predict the influence of different
ingredients, processing operations, and storage conditions on the stability and prop-
erties of food emulsions (McClements 2005b). In this book, the physical and chemi-
cal properties of emulsions are described with a special focus on food emulsions
stability. The experimental techniques for monitoring emulsion stability, the major
factors that influence the different destabilization mechanisms, as well as methods
of controlling them are discussed.
References
Corkery RW, Blute IA, Friberg SE, Guo R (2010) Emulsion inversion in the PIT range: quantita-
tive phase variations in a two-phase emulsion. J Chem Eng Data 55:44714475
Corredig M, Alexander M (2008) Food emulsions studied by DWS: recent advances. Trends Food
Sci Technol 19:6775
Davies E, Dickinson E, Bee R (2000) Shear stability of sodium caseinate emulsions containing
monoglyceride and triglyceride crystals. Food Hydrocolloids 14:145153
Fredrick E, Walstra P, Dewettinck K (2010) Factors governing partial coalescence in oil-in-water
emulsions. Adv Colloid Interface Sci 153:3042
Giermanska J, Thivilliers F, Backov R, Schmitt V, Drelon N, Leal-Calderon F (2007) Gelling of
oil-in-water emulsions comprising crystallized droplets. Langmuir 23:47924799
Giermanska-Kahn J, Laine V, Arditty S, Schmitt V, Leal-Calderon F (2005) Particle-stabilized
emulsions comprised of solid droplets. Langmuir 21:43164323
Golemanov K, Tcholakova S, Denkov ND, Gurkov T (2006) Selection of surfactants for stable
paraffin-in-water dispersions, undergoing solid-liquid transition of the dispersed particles.
Langmuir 22:35603569
Li Y, Le Maux S, Xiao H, McClements DJ (2009) Emulsion-based delivery systems for tributyrin,
a potential colon cancer preventative agent. J Agric Food Chem 57:92439249
Liu Y, Friberg SE (2010) Perspectives of phase changes and reversibility on a case of emulsion
inversion. Langmuir 26:1578615793
Mason TG, Wilking JN, Meleson K, Chang CB, Graves SM (2006) Nanoemulsions: formation,
structure, and physical properties. J Phys Condens Matter 18:R635R666
McClements DJ (2005a) Context and background. In: Food emulsions, principles, practices, and
techniques, 2nd edn. CRC Press, New York, pp 126
McClements DJ (2005b) Emulsion stability. In: Food emulsions, principles, practices, and tech-
niques, 2nd edn. CRC Press, New York, pp 269339
McClements DJ, Li Y (2010) Structured emulsion-based delivery systems: controlling the diges-
tion and release of lipophilic food components. Adv Colloid Interface Sci 159:213228
Relkin P, Sourdet S (2005) Factors affecting fat droplet aggregation in whipped frozen protein-
stabilized emulsions. Food Hydrocolloids 19:503511
6 1 Introduction
Sonneville-Aubrun O, Simonnet JT, LAlloret F (2004) Nanoemulsions: a new vehicle for skincare
products. Adv Colloid Interface Sci 108:145149
Thivilliers F, Drelon N, Schmitt V, Leal-Calderon F (2006) Bicontinuous emulsion gels induced by
partial coalescence: kinetics and mechanism. Europhys Lett 76:332338
Thivilliers F, Laurichesse E, Saadaoui H, Leal-Calderon F, Schmitt V (2008) Thermally induced
gelling of oil-in-water emulsions comprising partially crystallized droplets: the impact of inter-
facial crystals. Langmuir 24:1336413375
Thivilliers-Arvis F, Laurichesse E, Schmitt V, Leal-Calderon F (2010) Shear-induced instabilities
in oil-in-water emulsions comprising partially crystallized droplets. Langmuir
26:1678216790
van Aken GA (2001) Aeration of emulsions by whipping. Colloids Surf A Physicochem Eng Asp
190:333354
Vanapalli SA, Coupland JN (2001) Emulsions under shear. The formation and properties of par-
tially coalesced lipid structures. Food Hydrocolloids 15:507512
Vanapalli SA, Palanuwech J, Coupland JN (2002) Stability of emulsions to dispersed phase crys-
tallization: effect of oil type, dispersed phase volume fraction, and cooling rate. Colloids Surf
A Physicochem Eng Asp 204:227237
Walstra P (2003) Crystallization of lipids. In: Physical chemistry of foods. Marcel Dekker, New
York, pp 476547
Wooster TJ, Golding M, Sanguansri P (2008) Impact of oil type on nanoemulsion formation and
Ostwald ripening stability. Langmuir 24:1275812765
Chapter 2
Nano and Micro Food Emulsions
2.1 Methods of Formation
Emulsions with microdroplets, sometimes called conventional emulsions, and

nanodispersions, or thermodynamically stable emulsions (surprisingly called micro-
emulsions), can be easily manufactured on an industrial scale up. Due to their satis-
factory stability over a certain storage time and high bioavailability, they have
attained particular interest as delivery systems for bioactive substances, such as
carotenoids, phytostetol, polyunsaturated fatty acids, g-oryzanol, lipophilic vita-
mins, and numerous other compounds. Garti and co-workers (Amar et al. 2003;
Spernath et al. 2002), for example, prepared food-grade conventional emulsions
containing carotenoids with considerable success. Recently, studies have shown the
successful approach of using nanoemulsions to improve stability in food applica-
tions. Tan (2005) and Yuan et al. (2008) prepared b-carotene nanodispersions using
high-pressure homogenization and studied their physicochemical properties. Other
applications include the encapsulation of limonene, lutein, omega-3 fatty acids,
astaxantin, and lycopene (Chen et al. 2006), the encapsulation of a-tocopherol to
reduce lipid oxidation in fish oil (Weiss et al. 2006), and the use of nanoemulsions
to incorporate essential oils, oleoresins, and oil-based natural flavors into food prod-
ucts such as carbonated beverages and salad dressings (Ochomogo and Monsalve-
Gonzalez 2009).
2.1.1 Nanoemulsions
In the last two decades, nanotechnology has rapidly emerged as one of the most
promising and attractive research fields. The technology offers the potential to sig-
nificantly improve the solubility and bioavailability of many functional ingredients.
The high hydrophobicity of some bioactive substances makes them insoluble in
aqueous systems, and they therefore have a poor intake in the body. To improve

8 2 Nano and Micro Food Emulsions
carotenoids dispersibility in water, for example (Horn and Rieger 2001), and also
to increase their bioavailability during gastrointestinal passage (Deming and Erdman
1999), carotenoid crystals must be formulated, that is, to incorporate them in the
fine particles of oil-in-water (O/W) emulsions.
Nanoemulsions are nonequilibrium systems and cannot be formed spontane-
ously. They can be produced using two different approaches: high-energy and low-
energy methods. High-energy methods use intense mechanical forces to break up
macroscopic phases or droplets into smaller droplets and typically involve the use
of mechanical devices known as homogenizers, which may use high-shear mixing,
high-pressure homogenization, or ultrasonification. In contrast, low-energy meth-
ods rely on the spontaneous formation of emulsions under specific system composi-
tions or environmental conditions as a result of changes in interfacial properties
(McClements and Li 2010). Spontaneous emulsification is a less expensive and
energy-efficient alternative that takes advantage of the chemical energy stored in the
system (Bilbao Sinz et al. 2010). High-energy methods are effective in reducing
droplet sizes but may not be suitable for some unstable molecules, such as proteins
or peptides.
One of the most used low-energy methods is the phase inversion temperature
(PIT) method, which is based on the changes in solubility of polyoxyethylene-type
nonionic surfactants with temperature. The surfactant is hydrophilic at low tempera-
tures but becomes lipophilic with increasing temperature due to dehydration of the
polyoxyethylene chains. At low temperatures, the surfactant monolayer has a large
positive spontaneous curvature forming oil-swollen micellar solution phases (or
O/W microemulsions), which may coexist with an excess oil phase. At high tem-
peratures, the spontaneous curvature becomes negative and water-swollen reverse
micelles (or W/O microemulsions) coexist with an excess water phase. At a critical
temperaturethe hydrophiliclipophilic balance (HLB) temperaturethe sponta-
neous curvature is zero and a bicontinuous microemulsion phase containing compa-
rable amounts of water and oil phases coexists with both excess water and oil phases.
The PIT emulsification method takes advantage of the extremely low interfacial ten-
sions at the HLB temperature to promote emulsification. However, the coalescence
rate is extremely fast and the emulsions are very unstable even though emulsifica-
tion is spontaneous at the HLB temperature. By rapidly cooling or heating the emul-
sions prepared at the HLB temperature, kinetically stable emulsions (O/W or W/O,
respectively) can be produced with a very small droplet size and narrow size distri-
bution. If the cooling or heating process is not fast, coalescence predominates and
polydispersed coarse emulsions are formed (Ee et al. 2008).
Other low-energy methods that can be used to prepare nanoemulsions are the PIC
(phase inversion composition) method, in which the temperature is maintained con-
stant and the composition is changed (the solvent quality is changed by mixing two
partially miscible phases together). By using the PIC method, different nanomateri-
als such as colloidosomes (Dinsmore et al. 2002), cubosomes (Spicer 2004), and
microfluidic channels (Xu et al. 2005) have been prepared. Liu et al. (2006) used
polyoxyethylene (PEO) nonionic surfactants for the preparation of paraffin oil-in-
water nanoemulsions also by using the PIC method. They reported the preparation of
2.1 Methods of Formation 9
stable nanoemulsions with diameters ranging from 100 to 200 nm. Surfactants used
in this system were a combination of Span 80, a sorbitan monooleate with a low HLB
(4.3), and Tween 80, an ethoxilated sorbitan monooelate ester with a high HLB. As
these two surfactants possess the same backbone, they can mix easily, leading to a
controlled change in the final HLB.
Low-energy emulsification also includes the catastrophic inversion method
(CPI). In a typical phase inversion process, emulsification starts with a given emul-
sion morphology that inverts to an opposite emulsion by variations in emulsion
properties. For example, an oil-in-water system (O/W) inverts into a water-in-oil
system (W/O) and vice versa. Catastrophic inversion is induced by increasing the
volume fraction of the dispersed phase. In CPI emulsification, the system usually
begins with abnormal emulsions, that is, emulsions in which the surfactant has a
high affinity to the dispersed phase. Abnormal emulsions are usually unstable and
can only be maintained under vigorous mixing for a short period of time. The ulti-
mate fate of an unstable emulsion is to invert to the opposite state. A CPI is triggered
by increasing the rate of droplet coalescence so that the balance between the rate of
coalescence and rupture cannot be maintained. This may be induced by changing
the variables that increase the rate of droplet coalescence, such as the continuous
addition of dispersed-phase volume, the most common variable used, or by adding
a surfactant or a salt, or by altering the emulsification temperature or any parameter
that can significantly enhance droplets coalescence. Droplets formed via CPI are
usually above micrometer. Submicrometer droplets may only form if CPI of abnor-
mal to normal emulsion occurs in the vicinity of the locus of ultralow interfacial
tension (Sajjadi et al. 2004; Jahanzad et al. 2010). Peng et al. (2010) adopted a low-
energy method combining the PIT and CPI methods to prepare the water-in-oil
nanoemulsion. The aim of their work was to gain a better understanding of the rela-
tionship among the ratio of surfactants, the water/oil ratio, and long-term stability.
They found that the addition of a second surfactant to the formulation could provide
more stable nanoemulsions with the minimum size than only one surfactant. This
result was in agreement with other authors findings, who reported that to form
nanoemulsions, surfactant mixtures generally perform better than pure surfactants
for various applications (Rees et al. 1999; Uskokovi and Drofenik 2005; Pey et al.
2006). Peng et al. (2010) also reported the optimum composition for the systems
they studied.
Another commonly used inversion method is transitional phase inversion (TPI)
(Jahanzad et al. 2010). Before TPI can occur, the surfactants in both phases must
diffuse toward the interface, adsorb at the interface, and conform into a mixed sur-
factant layer at the optimum conditions. The rate of diffusion of the surfactants
depends on many parameters, including their size, the viscosity of the phase, and
the intensity of mixing, etc. For oil-in-water emulsions containing a pair of water-
soluble and oil-soluble surfactants, it was found that the addition of the water phase
containing the water-soluble surfactant to the oil phase containing the oil-soluble
surfactant may produce very fine emulsions if it is associated with interfacial ten-
sion lowering in the course of addition. The rate of addition of the second phase is
of great importance in achieving an emulsion with the desired properties. This is
because the dynamic of phase inversion emulsification is very fast, and the emulsion
properties change quickly with further addition, contrary to some conventional
emulsification methods. Therefore, it is important to find and maintain a semi-
equilibrated state in the course of emulsification during which sufficient surfactant
diffusion/adsorption can occur, and thus droplet rupturing is enhanced. TPI occurs
when the curvature of the oilwater interface gradually changes from positive to
negative, passing through a zero curvature at the inversion point. This is associated
with a shift in the surfactant nature from water-soluble to oil-soluble, or vice versa.
At the inversion point, the surfactant has a similar affinity toward both phases. As a
result, the interfacial tension passes through an ultralow value. This results in the
formation of emulsions with a very small drop size, sometimes called miniemul-
sions and nanoemulsions (Jahanzad et al. 2010).
In a recent work, Ribeiro et al. (2008) produced b-carotene-loaded nanodisper-
sions containing poly(D,L-lactic acid) (PLA) and poly(D,L-lactic-coglycolic acid)
(PLGA) by the solvent displacement method. Nanoparticles containing b-carotene
were produced by interfacial deposition of the biodegradable polymer, due to the
displacement of acetone from the dispersed phase. Gelatin or Tween 20 was used as
stabilizing hydrocolloids in the continuous phase. b-carotene was entrapped in the
polymeric matrix in the absence of any oily core material. In this kind of formula-
tion, polymers assumed the function of protective colloids and possibly also chemi-
cal stabilization of the nanodispersed phase.
2.1.2 Conventional Emulsions
Conventional emulsions, that is, emulsions with microdroplets, are thermodynami-

cally unstable liquidliquid dispersions. Emulsions with particle sizes higher than
100 nm are usually prepared by high-energy methods. As in the case of nanoemul-
sions, the O/W systems consist of lipid droplets dispersed in an aqueous medium,
with each lipid droplet being surrounded by a thin emulsifier layer. The initial droplet
concentration and size distribution of this type of delivery system can be controlled,
as can the nature of the emulsifier used to stabilize the droplets. A careful selection
of emulsifier type enables one to control interfacial properties such as charge, thick-
ness (dimensions), rheology, and response to environmental stresses (such as pH,
ionic strength, temperature, and enzyme activity) (McClements and Li 2010). To
form conventional emulsions, usually a preemulsion with coarse particles, obtained
by a mechanical device such as an Ultra-Turrax, is prepared. This system is further
homogenized to obtain a microdroplet emulsion. In general, a variety of homogeniz-
ers are available to prepare emulsions, including high-shear mixers, high-pressure
homogenizers, colloid mills, ultrasonic homogenizers, and membrane homogeniz-
ers. The choice of a particular kind of homogenizer and of the operating conditions
used depends on the characteristics of the materials being homogenized (e.g., viscos-
ity, interfacial tension, and shear sensitivity) and of the required final properties of
the emulsion (e.g., droplet concentration, droplet size, and viscosity) (McClements
2.2 Physical Chemical Properties 11
Fig. 2.1 Scheme of conventional emulsions preparation procedure
and Li 2010). Figure 2.1 illustrates a preparation procedure. This is a common

approach widely used in the literature. As an example, the typical conditions are as
follows: The fat and aqueous phases may be mixed using an Ultra-Turrax high-speed
blender operated at 20,000 rpm for 1 min. The resulting preemulsions may be further
homogenized for 20 min using an ultrasonic liquid processing. It is advisable that the
temperature of the sample cell is controlled by means of a water bath usually set at
a temperature that prevents protein denaturalization and that does not increase the
system viscosity very much, such as 15C. By doing this, the sample temperature
never rises higher than 40C during ultrasound treatment. Then emulsions are typi-
cally cooled quiescently to ambient temperature (22.5C) to perform physicochem-
ical analysis. Particle sizes obtained with this protocol may vary from 0.2 to 1 mm
depending on the system under study (lvarez Cerimedo et al. 2010).
2.2 Physical Chemical Properties
2.2.1 Nanoemulsions
The physical properties of nanoemulsions can be characterized by the combination

of a wide variety of techniques. For example, the macroscopic properties, such as
viscosity/viscoelasticity, conductivity, and interfacial tension, can be characterized
by rheometer, conductivity meter, and pendant drop tensiometer, respectively

(Boonme et al. 2006). The size and shape of the emulsion droplets were routinely
characterized by static and dynamic light scattering techniques (McClements 2005).
The major drawback of light scattering techniques is that dilution of emulsion sam-
ples is usually necessary to reduce multiple scattering and interdroplet interactions.
The dilution process may modify the structure and composition of the pseudoter-
nary phases of the nanoemulsions; therefore, the obtained results do not accurately
describe the actual system under study (Huang et al. 2010).
2.2.2 Conventional Emulsions
Conventional emulsions may also be characterized by the techniques mentioned above.

However, there are other alternatives that allow emulsions to be described without the
drawback of dilution. For example, particle size may be measured by low-resolution
nuclear magnetic resonance (NMR). As will be explained in Chap. 3, the principle of
this technique is completely different than that of light scattering techniques, and in
some cases, a more accurate description of the system may therefore be made.
2.3 Structuring Food Emulsions
The structure of the different pseudoternary phases can be investigated by small-

angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), and micros-
copy-like cryotransmission electron microscopy (TEM) (Spicer et al. 2001; Borne
et al. 2002; Boonme et al. 2006). These techniques may also be used to describe
conventional emulsions structure. By using SAXS measurements, lvarez
Cerimedo et al. (2010) proved that the role of trehalose in caseinate/fish oil emul-
sions went beyond the ability to form viscous solutions. For those systems, values
of q (the reciprocal lattice spacing, with q = 2p/d = 4p sin(q)/l, where d is the inter-
planar spacing and 2q is the Bragg angle) were significantly modified when the
aqueous phase contained trehalose compared to emulsions without sugar in the
aqueous phase. Values for emulsions with 10 wt.% fish oil and 5 wt.% sodium
caseinate were 241, 0.248, and 0.252 nm1 for emulsions with 0, 20, and 30 wt.%
trehalose, respectively. Some other aqueous phase components such as hydrocol-
loids proved to stabilize emulsions because they increase viscosity. The slightly
increased q values with the trehalose addition suggested that trehalose had an effect
beyond viscosity changes since modification of q values means that the aggregation
state of the protein changed with the aqueous phase formulation. These results were
in agreement with the small particle size as measured by dynamic light scattering
found when trehalose was added to aqueous phase. These results were corroborated
by confocal laser scanning microscopy (CLSM). CLSM is a more common technique
to describe emulsion structure. It is widely used in food applications and provides a
good description of the spatial distribution of different phases.
References 13
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Chapter 3
Methods for Stability Studies
The efficient development and production of high-quality emulsion-based products

depend on knowledge of their physicochemical properties and stability. A wide
variety of different analytical techniques and methodologies have been developed to
characterize the properties of food emulsions. Analytical instruments and experi-
mental methodologies are needed for research and development purposes to eluci-
date the relationship between droplet characteristics and the bulk physicochemical
and sensory properties of food emulsions, such as stability, texture, flavor, and
appearance. They are also needed in quality control laboratories and in food produc-
tion factories to monitor food emulsions and their components before, during, and
after production so as to ensure that their properties conform to predefined quality
criteria and/or to predict how the final product will behave during storage. This
chapter describes the most commonly used methods for stability studies, with a
focus on conventional food emulsions. Some of these techniques are also used in
nanoemulsions. Several examples of applications are described in detail.
3.1 Visual Observation
Emulsion systems have minimal thermodynamic stability and tend to phase-separate.

The primary driving force for phase separation is droplet interfacial free energy. The
inclusion of a surface-active substance that concentrates at the oilwater interface
imparts a degree of stability to these systems by lowering the interfacial tension. In
addition, a reduction in the interfacial tension facilitates emulsion formation and
prevents immediate droplet recoalescence during preparation. Visual observation is
an old method still used to analyze emulsion stability. Emulsion instability is stud-
ied by placing the samples in tubes, which are stored in quiescent conditions.
Destabilization is commonly indicated by the separation of water phase at the bot-
tom of the container or by a complete breakdown into two phases with a layer of
surfactant in between; that is, there is an opaque layer at the top, a turbid layer in the

16 3 Methods for Stability Studies
Fig. 3.1 Visual aspect of emulsions formulated with 0.3 wt.% of locust bean gum in the aqueous
phase and 10 wt.% of sunflower oil as the fat phase stabilized with different concentrations
of sodium caseinate (NaCas) and stored in quiescent conditions for a week at 22.5C. From left
to right: coarse emulsion stabilized with 0.5 wt.% NaCas; fine emulsion stabilized with 0.5 wt.%
NaCas; coarse emulsion stabilized with 2 wt.% NaCas; fine emulsion stabilized with 2 wt.%
NaCas; coarse emulsion stabilized with 5 wt.% NaCas; and fine emulsion stabilized with 5 wt.%
middle, and/or a transparent layer at the bottom. The serum layer may be defined as
the sum of the turbid and transparent layers (Mun et al. 2008), and creaming kinet-
ics may be followed by measuring the total height of the emulsion (HE) and the
height of the serum layer (HS). The extent of creaming may be characterized by a
creaming index as follows:
CI = 100 (HS / HE )
The creaming index is an indirect indication of the extent of droplet aggregation

in an emulsion (Mun et al. 2008). Figure 3.1 shows sample emulsions with different
creaming indexes.
Although visual observation provides limited information about food emulsion
stability, there are still new studies in the literature that are a contribution to their
fields. Some of them are mentioned here as an example.
Emulsifiers, usually small molecule surfactants, are added to the system to slow
emulsion-breaking mechanisms such as creaming and coalescence. The use of poly-
meric surfactants as emulsifiers and stabilizers in emulsions has attracted much
attention in recent years. Among the polymeric surfactants, hydrophobically modi-
fied water-soluble polymers have been a target of extensive studies because of their
potential industrial applications such as waterborne paints, coating fluids, cosmetics,
foodstuff, drug delivery systems, oil recovery, and water treatment and also because
of their relevance to biological macromolecular systems. This kind of polymer is a
3.1 Visual Observation 17
water-soluble polymer with a few hydrophobic groups in the hydrophilic macromo-

lecular chain. They can lower the surface and interfacial tension due to their intrinsic
amphiphilic properties, though their abilities are much lower than those of the con-
ventional low-molecular-weight surfactants. However, their solutions showed much
better thickening ability than low-molecular-weight surfactants due to their large
molecular structures and the association of the hydrophobic groups in the backbone.
Hence, the emulsification mechanisms differ from those of the low-molecular-weight
surfactants. Sun et al. (2007) studied the stability of emulsions formulated with a
hydrophobically modified hydroxyethyl cellulose (HMHEC) and paraffin oil. The
stability and droplet size distribution were investigated by visual observation, photo-
micrograph, and a laser scattering particle size distribution analyzer. Then the emul-
sions were transferred to a glass tube with a diameter of 1.6 cm and length of 15 cm
to monitor their stability to creaming and coalescence. The stability of emulsion with
time was accessed by monitoring the variation of the emulsion volume. The adsorp-
tion of HMHEC at the oilwater interface and the surface of emulsion droplets due
to the penetration of the alkyl chains in HMHEC into the oil phase was confirmed by
visual observation, the interfacial tension method, and an in situ environmental scan-
ning electron microscope (ESEM). According to the authors, the stability of emul-
sions prepared using HMHEC is based on both an associative thickening mechanism
caused by alkyl chains in HMHEC and the adsorption of HMHEC at the oilwater
interface, which can form a solid film, preventing coalescence of the droplets.
Proteins and polysaccharides are the two most important biopolymers used in
food emulsions to control their texture, microstructure, and stability. Polysaccharides
are mainly added to enhance the viscosity or to stabilize the system by the formation
of a gel, while proteins can form networks and have emulsification and foaming
properties (Dickinson 2003). Network formation and the phase properties of mixed
biopolymers affect the rheology of the system; therefore, a considerable research
effort has been devoted to understanding the mechanisms of network formation and
phase separation. Khalloufi et al. (2008) studied the behavior of two varieties of flax-
seed gums when added to whey protein isolate (WPI)-coated emulsion droplets at
neutral pH. Flaxseeds (Linum usitatissimum) have received increasing attention
because of their recognized health benefits, mainly related to the high level of w-3
fatty acids in the oil. A gum extract is obtained as the byproduct of the oil extraction,
and although quite rich in functional components, it is usually processed as livestock
feed. Flaxseed gum consists of a mixture of water-soluble polysaccharides with a
composition that varies depending on the extraction conditions. It is composed
mainly of L-galactose, D-xylose, L-rhamnose, and D-galacturonic acid. Concen-
trations ranging from 0% to 0.33% (w/v) of gum were added to the emulsions at pH 7.
At low concentrations [gum 0.075% (w/v)], no visual phase separation was
observed. However, at high concentrations of gum (0.1 w/v), there was visual phase
separation over time. Khalloufi et al. (2008) demonstrated that flaxseed gum is a non-
interacting polysaccharide at neutral pH; it could then be employed to strengthen the
nutritional value of some milk-based drinks, but at limited concentrations. To further
investigate the mechanisms of network formation and phase separation in polysac-
charide/protein systems, Moschakis et al. (2010) analyzed whey protein-stabilized/
sunflower oil emulsions in the presence of polysaccharide mixtures, using chitosan

and gum arabic at different weight ratios, and examined emulsions behavior in terms
of their stability, microstructure, and functional properties. Stability was followed by
visual observation. Freshly prepared emulsions were poured into 5-mL glass tubes
(height = 75 mm, diameter = 9 mm) after preparation. Subsequently, the tubes were
sealed to prevent evaporation. The tubes were then inverted carefully several times to
ensure thorough mixing. The emulsion samples were stored quiescently at ambient
temperature and the movement of any creaming boundaries was followed with time.
The emulsions stability, properties, and microstructure were found to be greatly
dependent on the precise gum arabic-to-chitosan ratio. Mixing of gum arabic with
chitosan led to the formation of coacervates of a size dependent on their ratio. The
incorporation of low gum arabic-to-chitosan weight ratios into emulsions caused
depletion flocculation and gravity-induced phase separation. Increasing the polysac-
charide weight ratio further generated a droplet network with a rather high viscosity
(at low-shear stress), which prevented or even inhibited phase separation. At even
higher gum arabic-to-chitosan ratios, the emulsion droplets were immobilized into
clusters of an insoluble ternary matrix.
The addition of the polymer to a dispersion can promote stability or destabilize
the dispersion, depending on the nature of interactions between the polymer and the
solvent and between the polymer and the dispersed particles (Hiemenz and
Rajagopalan 1997). The previous example and other studies with caseins that were
reported in the literature provide experimental support to Hiemenz and Rajagopalans
theory. According to this theory, usually called the polymer theory, some of the pos-
sible effects of polymer chains on a dispersion may be summarized as follows: In
the case of very low polymer concentrations, bridging flocculation may occur as a
polymer chain forms bridges by adsorbing on more than one particle. At higher
concentrations, distances among particles may be higher and mask van der Waals
attraction between the particles, causing steric stabilization. At moderate to high
polymer concentrations, the free polymer chains in the solution cause depletion
flocculation. At even higher polymer concentrations, the effect is known as deple-
tion stabilization. The polymer-depleted regions between the particles can only be
created by demixing the polymer chains and solvent. In good solvents, the demixing
process is thermodynamically unfavorable, and under such conditions one can have
depletion stabilization (Hiemenz and Rajagopalan 1997).
Today the food industry has a growing interest in the replacement of synthetic
emulsifiers by natural ones, such as polysaccharides and proteins. Among proteins,
the caseins, a group of unique milk-specific proteins that represent around 80% of
the total protein in the milk of cattle and other commercial dairying species, are the
most studied systems. Casein is widely used as an ingredient in the food industry;
its functional properties include emulsification, water-binding, fat-binding, thick-
ening, and gelation. Depending on the product, the caseinate content can range
anywhere from <1% to >50%. Casein is of particular importance as an emulsifier
because of its ability to rapidly confer a low interfacial tension during emulsifica-
tion and because of the strongly amphiphilic characteristics of the major individual
caseins. Casein in milk is strongly aggregated into polydispersed protein particles
3.1 Visual Observation 19
called casein micelles. The casein micelles are colloidal particles of self-associated
casein held together by nanometer-sized clusters of calcium phosphate and steri-
cally stabilized by an outer layer of the glycoprotein k-casein. Early electron
microscopy studies showed that the micelles have an uneven, raspberry-like appear-
ance, which was interpreted to mean that the micelles are built up from submi-
celles. However, in recent studies, the irregularities were considered to be
microtubules (Dalgleish et al. 2004). The micelles may be dispersed by adding a
calcium chelator or also by urea, SDS, high pH, or ethanol, indicating that hydro-
gen bonds, hydrophobic interactions, and electrostatic interactions are also involved
in micelle integrity. The caseins have been described as rheomorphic proteins,
indicating that they adopt molecular structures in solution dictated by the local
environment; that is, their structures are flexible enough to go with the flow.
Removing the calcium salts from milk casein and replacing them with sodium salts
leads to the production of sodium caseinate (NaCas). Commercial NaCas is a
variable multicomponent mixture containing four major constituents, as1, as2, b,
and k-casein. NaCas is composed of a soluble mixture of disordered hydrophobic
proteins having a strong tendency to associate into small protein particles (casein
submicelles, 2.5 10 5 Da ), which coexist in equilibrium with the free casein
molecules ( 2.5 10 4 Da ). In the casein micelle system, the micelle state may be
the lowest free-energy state of the system (Fox and Brodkorb 2008). Of particular
interest will be micelle structure and the mechanisms that operate in determining
micelle size since they are closely related to emulsion stability. Selecting sodium
caseinate as a stabilizer, Matsumiya et al. (2011) studied how the structural rela-
tionship between oil molecules and fatty acid residues of emulsifiers affects the
stability of its emulsions. Sodium caseinatebased emulsions were formulated
with seven different oils (corn oil, soybean oil, and five hydrocarbons, C10H22,
C12H26, C14H30, C16H34, C18H38) and diglycerol monoesters of different satu-
rated fatty acids or one mono-unsaturated fatty acid (lauric, myristic, palmitic,
stearic, or oleic acid). Emulsion destabilization, such as creaming, coalescence, or
phase separation, caused by diglycerol esters of fatty acids was visually observed.
These destabilization phenomena were recorded by taking pictures from 0 min
(just after adding emulsifiers) to 60 min. According to these authors, the molecular
structural similarity between dispersed oil molecules and emulsifiers, i.e., the simi-
larity of carbon chain length between hydrocarbon oil molecules and fatty acid
residues of emulsifiers, could affect the emulsion stability.
Collagen is a protein widely used in food industries to improve the elasticity,
consistency, and stability of foods, but this use has only been carried out in an
empirical way. A number of ingredients are obtained from collagen, such as gelatin
and collagen fiber. Collagen fiber is a new ingredient obtained from collagen in its
crude form, while gelatin is produced from the hydrolysis of collagen under drastic
conditions. Gelatin is widely used as a gelling agent, but it shows weaker emulsify-
ing properties than other surface-active biopolymers such as globular proteins. In
this context, de Castro Santana et al. (2012) investigated the effects of heat treat-
ment on the emulsifying properties of collagen fiber. First, the heat-treated collagen
fiber was characterized, and then the effect of this treatment, the pH condition, and
the homogenization process (rotor/stator device and high-pressure homogenizer)

were evaluated, determining emulsion properties such as phase separation, droplet
size, and rheology. To analyze the creaming stability, aliquots of the emulsions pre-
pared using the rotor/stator device and the high-pressure homogenizer were placed
in graduated 10-mL (diameter = 15.5 mm, height = 65 mm) and 50-mL (internal
diameter = 25 mm, height = 95 mm) test tubes. The larger cylinder used on fine
emulsions evaluation was chosen in order to reduce wall effects, since it is well
known that confining walls exert an extra retardation effect on a spherical particle
settling in a liquid (Chhabra et al. 2003). The stability of the emulsions against
creaming was evaluated during 7 days of storage at room temperature (25C) by
visually monitoring the development of separate phases. The creaming index (CI%)
of the emulsions was calculated as CI (% ) = (Vs / Vi ) 100 , where Vi represents the
initial volume of the emulsion and Vs the volume of the serum layer formed at the
bottom of the tube (Keowmaneechai and McClements 2002). According to de
Castro Santana et al. (2012), the alkaline emulsions showed lower kinetic stability,
since collagen fibers have a lower net charge (z-potential) at higher pH values,
decreasing the electrostatic stability process. Heat treatment slightly decreased the
protein charge and significantly reduced the insoluble protein content, suggesting a
decrease in the emulsifying properties of the collagen fiber.
3.2 Rheological Methods
The stability and rheological properties of emulsions are largely determined by the
interactions between the droplets. The nature and strength of the interactions are, in
turn, dependent on the structure and composition of the adsorbed layer at the oil
water interface. In food colloids, the stabilizing layer around emulsion droplets is
compositionally and structurally complex. This obviously makes it difficult to dis-
entangle the relationship between the colloidal interactions and the macroscopic
emulsion properties (Dickinson 1998). One of the most important macroscopic
properties of a food emulsion is its rheological properties. Texture and mouth-feel
are of particular practical consequence, and the careful manipulation of emulsion
rheology over a range of conditions is essential to provide a successful, marketable
product. Food oil-in-water emulsions can range from mobile Newtonian liquids
such as milk to highly non-Newtonian viscoelastic products such as mayonnaise.
The rheological properties of emulsions are determined by many different factors,
including the concentration of the dispersed phase, the emulsion stability, the tem-
perature, and the viscosity (and composition) of the continuous phase. Manipulation
of these various parameters can be influential in determining the rheological charac-
teristics of an individual food emulsion product. Dickinson and Golding (1997a)
proved that there was a close relationship between the rheological behavior of casein
emulsions and protein concentrations using both steady-state viscometry and small-
deformation oscillatory methods. That study demonstrated that small-deformation
rheometry was a suitable tool for probing emulsion instability mechanisms that can-
not readily be determined from visual or ultrasound creaming observations alone.
3.2 Rheological Methods 21
3.2.1 Small-Deformation Rheology
Interfacial rheology has been used to quantify the film strength of adsorbed emulsi-
fier layers at fluid interfaces. There are two types of interfacial rheology: dilatational
and shear. Dilatational techniques involve inducing a change in the interfacial area
while simultaneously measuring the interfacial tension. Shear methods involve
inducing shear in the film without a change in area; many different arrangements
exist for this type of measurement in particular that allow measuring both mechani-
cal (solid-like) and flow (liquid-like) properties of emulsions. Values of interfacial
tension are very relevant to emulsion stability. However, for long-term emulsion
stability to coalescence and hence phase separation, the strength of the interfacial
film formed by a prosurface-active substance has been reported to be more impor-
tant than its effect on interfacial tension (Opawale and Burgess 1998). Proteins have
a polymeric and polyelectrolyte nature. Colloidal stabilization by protein adsorption
is frequently the result of a combination of steric and electrostatic (electrosteric)
repulsion. These forces originated for the adsorbed protein film impact interfacial
rheology.
The rheological properties of oil-in-water (O/W) emulsions are generally con-
trolled by varying the droplet volume fraction, the droplet size distribution, and,
mainly, the interdroplet forces by additives. Following time-dependent changes on
rheological properties of emulsions with a high oil-to-protein ratio (5.835),
Dickinson and Golding reported in an early work (1997a) that the rheological
behavior of caseinate emulsions was largely determined by the interactions between
droplets and especially by the nature and strength of the interparticle attractive
forces, which were dependent on the structure and composition of the adsorbed
layer at the oilwater interface. The aim of that work was to provide experimental
evidence in support of polymer theory. Their findings may be summarized as
follows: The stability and rheology of emulsions made with NaCas depend on
two main factors, the structure and composition of the adsorbed protein layer at the
oilwater interface, and the state of self-assembly and aggregation of the protein in
the aqueous phase. In emulsions containing protein at concentrations well below
that required for monolayer saturation coverage, the system exhibits time-dependent
bridging flocculation and coalescence. At protein contents around that required for
saturation monolayer coverage, the system is Newtonian and unflocculated and is
very stable toward creaming and coalescence. At higher protein contents, the cream-
ing stability of the pseudo-plastic system is greatly reduced due to depletion floc-
culation of protein-coated droplets by unadsorbed submicellar caseinate. At even
higher protein contents, there is partial restabilization of the flocculated emulsion in
the form of a strong particle gel network. At low concentrations of protein, no floc-
culation was observed immediately after preparation, but the rheological behavior
with time showed that there was a steady increase in droplet aggregation. The
35 vol.% n-tetradecane emulsions with protein concentration of 5 or 6% wt.% had
not only an initial flocculation more extensive than emulsions with 14% wt.% pro-
tein but also showed a higher extent of structural rearrangement during aging.
According to the authors, rearrangement to more closely packed structures would
therefore tend to result in a greater reduction in apparent viscosity as more of the

trapped continuous phase is released into the bulk. The rheological behavior of their
systems demonstrated that the droplet networking effect was very relevant since
with the casein systems even up to protein concentrations of 8 wt.%, there was rela-
tively little contribution to the emulsion rheology from the viscoelastic properties of
the continuous phase. So the structural mechanism influencing the rheology of the
casein-rich emulsions can apparently be considered entirely attributable to inter-
droplet depletion interactions.
Schokker and Dalgleish (1998) suggested that the functional properties of the
casein layer at the oilwater interface could be related to the flocculation behavior.
In the 20 vol.% soybean oil, 1 wt.% sodium caseinate aqueous solution emulsions
they studied, the systems were more susceptible to shear-induced flocculation after
preparation than during storage. However, no proteolysis products could be detected
by SDS-PAGE, and also no exchange of protein between the droplet surface and the
continuous phase was observed in the stored emulsions.
Stevenson et al. (1997) found that displacement of b-casein from the oilwater
interface with Tween 20 was more difficult after storage at room temperature, pre-
sumably caused by rearrangements of molecules at the interface and an increase in
hydrophobic interactions at the interface. A similar mechanism was proposed by
Schokker and Dalgleish (2000) to interpret the decreased susceptibility with time of
the emulsion to flocculate under shearing conditions.
Berli et al. (2002) proposed a theory to connect the interparticle forces and the
rheology in order to manipulate the colloidal stability of emulsions. In agreement
with Dickinson et al. (1997), they found that the rheological response of the emul-
sion was highly dependent on the concentration of caseinate. When the concentra-
tion of free proteins in the bulk solution was low, emulsions were Newtonian, while
for higher caseinate concentrations, emulsions became shear-thinning. According
to Berli et al. (2002), the variation of emulsion viscosity with protein concentration
could not be explained by the variation in the suspending fluid viscosity. The strength
of interparticle interactions and a genuine variation in the radius of the submicelle
with formulation had a key role in rheological behavior.
In a recent work, Tipvarakarnkoon et al. (2010) studied the stabilizing effect of
modified acacia gum on the stabilization of coconut O/W emulsions. For this pur-
pose they prepared model substrates for coconut milk or coconut cream. Coconut
milk is widely used as a food ingredient not only in Southeast Asia but also in
Western countries. It is an O/W emulsion of varying fat contents, prepared from
coconut meat by extraction. The native coconut proteins of the milk are not able to
stabilize the emulsion sufficiently against creaming during storage. Therefore, vari-
ous methods for coconut milk stabilization have been tested. On the one hand, dif-
ferent emulsifiers such as surfactants (Tween 20, SDS) as well as milk proteins
(sodium caseinate) or whey protein isolate have been used (Jena and Das 2006;
Tangsuphoom and Coupland 2008, 2009a, b). On the other hand, stabilizing carbo-
hydrates such as coconut sugars or carboxymethylcellulose have been added
(Jirapeangtong et al. 2008) alone or together with emulsifiers. Tipvarakarnkoon
et al. (2010) used acacia gum since it has emulsifying as well as stabilizing abilities.
This native glycoprotein is well known and extensively used as an emulsifier and
stabilizer in the food industry. The properties of native acacia gum, however, differ
strongly in dependence of the raw material. Therefore, in this work it was modified
by a maturation process that increased the emulsifying arabinogalactan protein
share. All emulsions proved to be low-viscous and nearly Newtonian liquids. It can
be concluded that the emulsion stability was mainly a result of the excellent emul-
sifying properties and not of an additional thickening effect of the gums. The modi-
fied acacia gums can be recommended as an emulsifier and stabilizer for application
in different food products, preferably in low-viscous emulsions such as coconut
milk drinks or other beverages.
Mixtures of surface-active substances are often used in many technological
applications, including food and pharmaceutical industries, cosmetics, coating pro-
cesses, and so forth. In many of these applications, proteinsurfactant mixtures are
used in the manufacture of processed dispersions. These dispersions contain two or
more immiscible phases (aqueous, oil, and/or gas phases) in the form of foams and
emulsions. In food systems, the interfacial layer often comprises both proteins and
surfactants (mainly lipids and phospholipids). The optimum use of emulsifiers in
food technological applications depends on our knowledge of their interfacial phys-
icochemical characteristics, such as surface activity, amount adsorbed, the kinetics
of film formation, structure, thickness, topography, ability to desorb (stability), lat-
eral mobility, interactions between adsorbed molecules, ability to change conforma-
tion, interfacial rheological properties, etc. Maldonado-Valderrama and Rodrguez
Patino (2010) published a review focused on the interfacial rheology of protein
surfactant mixtures, putting more emphasis on the interfacial dilatational rheology.
According to the authors, literature reports has shown that interfacial rheology of
proteinsurfactant mixed films depends on the protein (random or globular), the
surfactant (water-soluble or oil-soluble surfactant, ionic or nonionic), the interface
(airwater or oilwater), the interfacial (protein/surfactant ratio) and bulk (i.e., pH,
ionic strength, etc.) compositions, the method of formation of the interfacial film
(by spreading or adsorption, either sequentially or simultaneously), the interactions
(hydrophobic and/or electrostatic), and the displacement of protein by surfactant.
Proteins and surfactants can form nano-scaled micelles and micro-scaled vesicular,
crystalline, or other structures in the bulk phase prior to adsorption at the interface
during dispersion formation. However, according to Maldonado-Valderrama and
Rodrguez Patino (2010), little is known about the effect of these nano- and micro-
structures on the interfacial rheology and dispersion characteristics. Moreover, the
relationship between the homogeneity/heterogeneity (i.e., aggregation, miscibility,
phase separation, etc.) of a fluid interface and the macroscopic properties of the
dispersion is only indirectly known from the rheological properties of the fluid
interface. More research should be performed in order to establish quantitative cor-
relations between the interfacial rheology (including the effect of pH, ionic strength,
temperature, high pressure, addition of food reagents, protein modifications, etc.)
and dispersion stability of pure and proteinsurfactant mixed systems.
Murray (2011) has reviewed the scientific literature from 2002 to the present on
the interfacial rheology of protein films, focusing on the implications for biological
systems and in particular for food emulsions and foams. The areas covered include
new methods of measurement; proteins and polysaccharides and protein/polysac-
charide complexes; the effects of cross-linking within protein films and the origins of
film viscoelasticity; proteins and low-molecular-weight surfactants; experimental
and theoretical studies of the interfacial rheology and its relationship to emulsion and
foam stability. According to Murray (2011), there has been a something of a resur-
gence of interest in these areas, resulting in a number of important advances that
should aid further understanding and exploitation of proteins as surface-active agents
and colloid stabilizers. As conclusions of his review, it may be mentioned that there
have been a number of important advances in the study of the interfacial rheology of
adsorbed protein films in the last 10 years or so. These have been most notable in
instrumentation and measurement techniques, but also in an improved understanding
of the behavior of proteins mixed with other surface-active agents or with molecules
that can form complexes with the proteins that affect their adsorption. The diverse
range of interfacial properties of proteins and other biosurfactants suggests that they
could be more widely used as effective but biocompatible, biodegradable colloid
stabilizers. Various new methods of measurement of the interfacial rheology of
adsorbed protein together with improvements in the sensitivity and analysis of tradi-
tional methods have been described in detail in the review (Murray 2011).
3.2.2 Viscosity
Viscosity increase is a common strategy to enhance emulsion stability. This goal

may be reached by adding compounds such as hydrocolloids or proteins. In this
section, well describe some examples of products in which viscosity is a main
property.
In the field of food additives, alcoholic cream liqueur emulsions are of special
interest. The stability and shelf life of these emulsions depend on several factors,
such as viscosity, volume particle size, temperature, pH, and ionic forces. The effect
of caseinates is commonly underestimated; for example, the content of Ca++ and Na+
in caseinates influences the stability of alcoholic emulsions. A commercial use of
caseinates in alcoholic cream liqueurs is as an emulsifying agent. Cream liqueurs of
about 15% alcohol content can be prepared with an extended shelf life of several
years. Medina-Torres et al. (2009) explored the effect of several kinds of commer-
cial caseinates and their mixtures into the stability and shelf life of alcoholic cream
liqueurs as estimated by rheological and volume particle size distribution behavior
at different storage times and temperatures. Alcoholic emulsions were prepared
with three different caseinate batches at 3% and 4% (w/v) protein content and 16.9%
(v/v) ethanol content. Two storage temperatures (25C and 40C) and three storage
times (0, 25, and 45 days) were used. The stability of the emulsion was followed
visually by detecting coalescence. The preparation of emulsion for viscosity mea-
surements was as follows: 70.3 g of a caseinate batch without alcohol (about 67.5 g
of dry matter) was weighed in a 600-mL beaker (weight A, g) and cold water
(379.7 g) was slowly added using a rotor/stator system at 300 rpm for 20 min at
25C. The mixture was stored for 12 h at room temperature to stabilize and to allow
for lumps to be moistened. Then the beaker with its contents was placed in a water
bath at 70C for 30 min under occasional stirring to keep a homogeneous solution.
After dissolving, the mixture was completed to the initial weight (A + 450 g) to
compensate for evaporated water, cooling the beaker under running water at 20C
for 4 h. The rheological measurements were performed on a strain-controlled rhe-
ometer using a double concentric geometry. The viscosity was measured at different
storage times and temperatures (0, 15, 30, and 45 days, 25C and 40C, respec-
tively). The protein content showed to be crucial to reduce coalescence. For all
emulsions with 4% protein content, coalescence visually appeared at a higher alco-
hol content than for emulsions with 3% protein content. The volumetric diameter
(d4,3) was a function of storage time, and in all cases the coalescence increased.
Viscosity also increased with storage time in all the blends. The viscosity was found
to be directly related to the particle size of the emulsions prepared at different
caseinate ratios. Casein emulsions with intermediate viscosity values were the most
stable. Emulsions having low viscosity values are an advantage in the elaboration of
liqueur creams because the formed micelles are more stable, avoiding the possible
aggregation or precipitate formation. Although a small initial average droplet size in
prolonged storage times does produce a short-term stability improvement, there was
not yet a significant effect on long-term product stability. Finally, the results on
microscopy suggested that the final structure plays an important role in the stability
of the alcoholic emulsion with industrial interest in food emulsions, particularly in
the elaboration of cream liqueurs.
Mango (Mangifera indica L.) is a tropical fruit in the plant family Anacadiaceae
that originated in India and Southeast Asia. The mango pulp contains vitamins and
bioactive compounds such as b-carotene and vitamins A, C, and E. Ripe mango
pulp has b-carotene of 50% of total carotenoid and 2.0% (w/w) pectin, which is a
soluble dietary fiber. In some countries, mango is mixed with milk to become cream
stuff used in dessert for mango flavor, taste, and homogeneous texture. This mango
cream is similar to custard, mayonnaise, and salad dressing, which are oil-in-water
emulsions. Proteins and polysaccharides are commonly used together in oil-in-
water food emulsions. Proteins are widely used as an emulsifier because they have
an ability to adsorb at the oilwater interface and stabilize the oil droplets, while
polysaccharides are usually added to increase the viscosity of emulsion (Dickinson
1995). Data in the literature indicated that both pectin and/or sodium caseinate had
an effect on emulsion stability. To further investigate these ideas, Karunasawat and
Anprung (2010) studied the effect of depolymerized mango pulp as a stabilizer in
oil-in-water emulsion containing sodium caseinate. The objective of that paper was
to investigate whether the degree of hydrolysis of mango pulp and the sodium
caseinate concentration have effects on the oil-in-water emulsion stability in terms
of average droplet size, viscosity, and creaming stability. The apparent viscosity of
systems was measured by using a controlled stress rheometer equipped with cone
and plate geometry. A cone diameter of 40 mm with a cone angle of 4 was used.
The emulsions viscosity was measured at room temperature (30C 1C) about
24 h after the emulsion was made. Increasing the degree of hydrolysis for pectin in
mango pulp resulted in a decrease in the emulsions viscosity. It can be seen that
there was no change in the viscosity of emulsions over the experiment. This can be
expected since the emulsions did not display flocculation or coalescence after a stor-
age period of 1 day. The results reported by Karunasawat and Anprung (2010) indi-
cated that the viscosity of the emulsions containing 0.5% or 1.0% (w/w) sodium
caseinate was related to the average droplet sizes. It might be expected that nonfloc-
culated samples behave as Newtonian fluids, while flocculated emulsions show
pseudo-plastic behavior. As expected, the higher sodium caseinate concentration
[>2.0% (w/w)] induced depletion flocculation in the emulsions. The entrapment of
a certain amount of continuous phase in the flocculated structure caused an increase
in the effective volume fraction of hydrodynamically interacting entities, which in
turn increased the viscosity of aggregated emulsion systems according to the
DoughertyKrieger equation (McClements 1999). However, the flocculated struc-
ture was broken up with storage time, and some trapped continuous phase was
released, consequently lowering the viscosity following the behavior described by
Dickinson and Golding (1997a, b). For the emulsions containing sugar content of
46 mg of glucose/g fresh weight (DP 46E pectinase treated or DP 46), there was a
decrease in viscosity with increasing shear rate, indicating shear-thinning (pseudo-
plastic) behavior of the emulsions containing DP 46E or DP 46. The emulsion with
the best stability was that made from depolymerized mango pulp with reducing
sugar content of 60 mg of glucose/g fresh weight (DP 60) and 2% (w/w) sodium
caseinate. In addition, it was found that DP 60 could be used as an alternative stabi-
lizer for oil-in-water food emulsions.
Alginate is a linear copolymer with homopolymeric blocks of (14)-linked b-D-
mannuronate (M) and its C-5 epimer a-L-guluronate (G) residues, respectively,
covalently linked together in different sequences or blocks. Alginate has a number
of free hydroxyl and carboxyl groups distributed along the backbone; therefore, it
may be functionalized by chemical modification. By forming alginate derivatives
through functionalizing available hydroxyl and carboxyl groups, properties such as
solubility and hydrophobicity and physicochemical and biological characteristics
may be modified (Yang et al. 2011). Alginates are widely used as a gelling agent for
thickening foods and cosmetics. Yang et al. (2012) modified alginate with the aim
of using it as an emulsifier in emulsions. The stability of their systems was followed
by visual observation, droplet size, microstructure, and viscosity. Viscosity mea-
surements were conducted in a rheometer using a cone-and-plate geometry, with a
cone angle of 1 and a diameter of 60 mm. The samples were introduced onto the
plate for 5 min to eliminate residual shear history and then experiments were carried
out immediately. The measuring device was equipped with a temperature unit that
gave good temperature control (25C 0.05C) over an extended time. Their studies
showed that the emulsions containing 1.0 wt.% sodium alginate and 0.3 and 0.5 wt.%
dodecanol alginate were unstable, whereas the emulsions containing 0.81.2 wt.%
dodecanol alginate presented better stability during storage.
It is well known that the polymersurfactant complexation in colloidal formula-
tions can alter their stability and rheology. Studies show that the stabilization of
3.3 Ultrasound Profiling 27
emulsions by using a combination of surfactant and polyelectrolytes (Stamkulov

et al. 2009) or solid nanoparticles (Binks et al. 2007) can synergistically enhance
their stability. Nambam and Philip (2012) investigated the competitive adsorption of
polymer and surfactant at an oilwater interface by measuring the hydrodynamic
diameter, z-potential, microstructure, and rheology. The polymer selected was a
statistical copolymer of polyvinyl alcohol and vinyl acetate copolymer (PVAVAc),
and the emulsion was an oil-in-water system with an average droplet diameter of
200 nm. The oil consisted of oleic acidstabilized Fe3O4 nanoparticles of size 10 nm
and the surfactant sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide
(CTAB), and nonylphenol ethoxylate (NP9). The questions they tried to address
from those studies were the following: What was the equilibrium situation of poly-
mersurfactant complexation when both the polymer and surfactant have abilities to
complex with each other and at the same time they can also adsorb at the droplet
interface independently? What is the consequence of such complexation on the sta-
bility (agglomeration) of emulsions? What are the conditions to minimize floccula-
tion of emulsion droplets? The enhanced viscosity upon the addition of ionic
surfactant into polymers confirms the strong interaction between them. Their stud-
ies show that lower-molecular-weight polymers with suitable ionic surfactants can
synergistically enhance the stability of formulations, while longer-chain polymers
induce bridging flocculation. Their results were useful for preparing oil-in-water
formulations with long-term stability.
3.3 Ultrasound Profiling
In a pioneering work, Dickinson and Golding (1997b) followed the stability of

emulsions with a high oil-to-protein ratio (5.835) by the technique of ultrasonic
velocity scanning. The bases of the technique are as follows: The velocity of sound
in a dispersion is related to the physical properties of dispersed and continuous
phases and the relative quantities of each phase. When a planar sound wave is inci-
dent on a spherical particle, the wave is scattered; that is, a proportion of the energy
is removed from the forward direction of the wave and is transmitted in other direc-
tions. The phase changes in the forward component of the wave manifest them-
selves as a change in the apparent velocity of the sound wave. Thus, the velocity
becomes a function of parameters such as particle size and the differences in physi-
cal properties between the two phases. Creaming of an emulsion occurs when the
dispersed phase is less dense than the continuous phase and so drifts to the top of the
sample, forming a cream layer. This method allows one to detect the creaming
destabilization of emulsion systems by recording the profiles of the dispersed-phase
concentration as a function of heights at different times. These measurements pro-
vide the means for stability studies through the use of an equation to relate the
ultrasound velocity to the dispersed-phase concentration, that is, the volume frac-
tion. The approach in each case depends on the systems under study and is usually
closely associated with the Urick equation (Pinfield et al. 1995). The model systems
studied by Dickinson and Golding (1997b) were formulated with NaCas (16 wt.%)
as the sole emulsifier, n-tetradecane as the fat phase (35 vol.%), and a phosphate
buffer as the aqueous phase. Their results showed that creaming and flocculation
kinetics had a complex dependence on caseinate content. The most stable caseinate
emulsions with respect to creaming were those for which there was sufficient pro-
tein present to give excellent surface coverage and associated steric/electrostatic
stabilization, but where the concentration of unbound protein was still low (below
the critical flocculation concentration) so that there was no depletion flocculation.
In an emulsion, the volume fraction of each phase is defined as the radio of the
volume of each phase over the total volume. The dispersed phases volume fraction
and droplet size distribution of emulsions determine many of their most important
physicochemical properties, including their stability, rheology, and appearance.
Usually, a high volume fraction for the dispersed phase induces an opaque emulsion
and prevents the utilization of light to analyze it. A way to circumvent this difficulty
of analyzing by light is the use of ultrasonics as an inquiry method. Polysaccharides
may interact with protein adsorbed at the interface. Depending on the size and
charge of the molecules, the concentration of polysaccharide present, and the envi-
ronmental conditions of the solution (pH and ionic strength), the proteinpolysac-
charide interactions can (1) improve the stability of the emulsion (may have an
associative interaction), (2) lead to destabilization by bridging flocculation, where a
long chain polymer is present in small concentrations and adsorbs onto more than
one colloidal particle, or depletion flocculation, where the intercolloidal region
becomes depleted of polymer, creating a polymer concentration gradient, hence
an osmotic pressure difference, which draws the particles closer to one another, or
(3) last, cause gelation. Understanding the assemblies occurring at the interface and
the dynamics of the interactions is fundamental for engineering food emulsions.
Ultrasonic spectroscopy has been employed to characterize dispersed systems such
as emulsions and milk and to study proteinpolysaccharide interactions (Dwyer
et al. 2005; Corredig et al. 2004a; Dalgleish et al. 2005). The ultrasonic waves have
the ability to propagate through optically opaque materials and therefore are highly
suited for use in concentrated colloidal systems. The parameters obtained are sensi-
tive to the molecular organization and molecular interactions in the samples.
Ultrasonic spectroscopy is therefore noninvasive and can determine changes in
physicochemical rearrangements in colloidal systems. In studies focusing on struc-
ture development, the stages preceding aggregation and the sol-to-gel transition are
of extreme importance, as these moments are critical for the physical and sensorial
characteristics of the final gel. With conventional techniques such as traditional
light scattering or rheology, information on the liquid-to-solid transition state might
be lost. Ultrasound spectroscopy also shows great potential for the study of in situ
changes in emulsion systems. This technique has been successfully employed to
study dropletdroplet interactions in situ to distinguish differences between bridg-
ing flocculation and depletion flocculation in whey proteinstabilized emulsions
(Gancz et al. 2005).
High-methoxyl pectins (HMP) are negatively charged polysaccharides com-
posed of a backbone of methyl-esterified galacturonic acid (>50% substituted) with
3.3 Ultrasound Profiling 29
branched regions containing arabinose, galactose, and xylose. Changes in charge

and charge distribution on the pectin molecule strongly affect the interactions with
proteins. Liu et al. (2007a) described the application of high-resolution ultrasonic
spectroscopy on the study of the acid-induced aggregation of sodium caseinate
emulsions in the presence of HMP. The effect of pH as well as the concentration and
charge (degree of esterification, DE) of HMP on the structural changes were
observed by measuring ultrasonic velocity and attenuation. They found that during
acidification, caused by the addition of glucono-d-lactone, there were small changes
in the overall ultrasonic velocity. Although small, it was possible to relate these
changes to the structural changes in the emulsion. The values of ultrasonic attenua-
tion decreased at high pH with increasing amount of HMP, indicating changes in the
flocculation state of the oil droplets caused by depletion forces. During acidification
at pH 5.4, emulsions containing HMP showed a steep increase in the ultrasonic
attenuation, and this pH corresponds to the pH of association of HMP with the
casein-covered oil droplets. The adsorption of HMP onto the interface causes a rear-
rangement of the oil droplets, and the emulsions containing sufficient amounts of
HMP no longer gel at acidic pH. This research demonstrated for the first time that
ultrasonic spectroscopy can be employed for in situ monitoring and analysis of acid-
induced destabilization of food emulsions.
One of the major disadvantages of applying the ultrasonic technique to emul-
sions is that a considerable amount of data about the thermophysical properties of
the component is needed to interpret ultrasonic spectra, such as adiabatic compress-
ibility, density, viscosity, conductivity, specific heat capacity, and cubical expansion
coefficient. Values of these properties for a variety of liquids have been compiled.
Nevertheless, all of these thermophysical properties are temperature-dependent,
which means that the ultrasonic properties of emulsions vary with temperature.
Chanamai et al. (1998) investigated the influence of temperature on the ultrasonic
velocity and attenuation coefficient of oil-in-water emulsions with different compo-
sitions. They also explored the implications of the temperature dependence of the
ultrasonic properties of emulsions for the utilization of ultrasonic techniques for
characterizing emulsions. Their results indicated that it is important to either care-
fully control or measure the temperature during an ultrasonic analysis. The mea-
sured ultrasonic properties of the emulsions were in reasonable agreement with
those predicted by ultrasonic scattering theory across most of the temperature range.
Nevertheless, there were some significant disagreements, especially at high tem-
peratures, which thus highlight the need for researchers to accurately measure and
compile the temperature dependence of the thermophysical and ultrasonic proper-
ties of a wide variety of different liquids.
A wide variety of experimental techniques have been developed in the past 3040
years to characterize the droplets in emulsions, including electron and light micros-
copy, dynamic and static light scattering, neutron scattering, and electrical conduc-
tivity measurements. Most of these methods cause some amount of disruption,
during either the analysis or sample preparation stages. This makes it difficult to
accurately analyze in detail the changes occurring during colloidal destabilization,
namely, solgel transitions or phase separations, as these mechanisms are dependent
on the volume fraction or minimal shear disturbance. In addition, most of these

techniques are either destructive or only suitable for application to dilute optically
transparent systems, whereas most emulsions of practical importance are concen-
trated and optically opaque. It was clear at that time that there was a need for ana-
lytical techniques to provide information about the physicochemical properties of
concentrated emulsions. Thus, for this purpose, new techniques such as NMR and
ultrasonic spectroscopy were developed. McClements and Coupland (1996) proved
the fitness of ultrasound spectroscopy for measuring droplet size distributions in
concentrated emulsions in situ. The ultrasonic velocity and attenuation coefficient
of an emulsion are measured over a range of frequencies, and then the multiple-
scattering theory is used to convert these measurements into a droplet size distribu-
tion. The theory they selected assumes that droplets are spherical, much smaller
than the ultrasonic wavelength, and do not physically interact with their neighbors
and that higher-order scattering terms are negligible. According to McClements and
Coupland (1996), the first two assumptions are valid for most emulsions. Droplets
only become nonspherical at high-volume fractions, in high-shear fields, or when
they crystallize. The majority of emulsion droplets of importance are between 0.1
and 10 mm, whereas the ultrasonic wavelength is greater than 150 mm at frequencies
of 10 MHz and less. The latter two assumptions may be violated in concentrated
emulsions or in systems where the droplets flocculate, and can lead to significant
deviations between experimental measurements and theoretical predictions. Despite
these limitations, the ultrasonic theory presented by McClements and Coupland
(1996) has been shown to be applicable to a number of oil-in-water emulsions up to
fairly high-volume fractions (2030%). One advantage of this technique is that it
can easily be adapted for online measurements, which could be of considerable
importance for many manufacturing processes. One of the major limitations of the
ultrasonic technique is that it cannot be used to study emulsions that contain small
gas bubbles. This is because the gas bubbles scatter the ultrasound so effectively
(even at very low concentrations) that the ultrasonic signal may be completely atten-
uated. Ultrasound also has limited application to very dilute emulsions (<0.5 wt.%)
because the change in ultrasonic properties with droplet size becomes of the same
order as the experimental error. In summary, ultrasonic spectroscopy is most useful
for studying concentrated or optically opaque materials, or when online measure-
ments are required. For dilute emulsions (<1%), light scattering techniques are usu-
ally preferable.
Glseren and Corredig (2011) reported that there are four major mechanisms that
contribute, generally additively, to the magnitude of attenuation in colloidal systems,
and they are dependent on the material properties: Intrinsic attenuation is a material
property and is observed in continuous phases as well as in dispersed phases; visco-
inertial losses are due to the density differences between the dispersed and the con-
tinuous phases; thermal losses are due to the temperature gradient between the
continuous and dispersed phases that is generated by the ultrasonic compression-
decompression cycles; and scattering losses take place as an ultrasound wave is
refracted, diffracted, and reflected by the dispersed phase particles. Increasing ultra-
sonic frequencies (i.e., decreasing wavelengths) enable us to investigate increasingly
3.4 Electroacoustic Spectroscopy: z-Potential 31
smaller size scales, and scattering attenuation becomes comparatively larger with
frequency. According to the authors, the primary loss mechanism in food emulsions
of a few mm in diameter is thermal losses, especially at frequencies lower than
100 MHz. In agreement with McClements and Coupland (1996), they concluded that
based on the material properties, the measured frequency dependence of attenuation
can be exploited to derive particle size distributions.
3.4 Electroacoustic Spectroscopy: z-Potential
Over the past two decades, electroacoustic (EA) techniques have emerged as a pow-
erful means of monitoring droplet charge (z-potential) in colloidal systems. As hap-
pens with ultrasonic spectroscopy, electroacoustic spectroscopy is a nondestructive
technique. It also has the potential to analyze concentrated colloids or emulsions
online and in situ and does not suffer from opacity of samples, a major problem for
most light scattering techniques. Thus, the major advantage of the EA technique
over more conventional microelectrophoretic techniques based on light scattering is
that it is capable of analyzing emulsions with high droplet concentrations (<50%)
without any sample dilution. EA spectroscopy can also be used to provide informa-
tion about the droplet size distribution of emulsions; however, the droplet size range
is usually rather limited (0.110 mm). Nevertheless, there are some limitations of
the EA technique for certain applications. For example, the droplets must have an
electrical charge, there must be a significant density contrast (Dr > 2%) between the
droplets and the surrounding liquid, and the viscosity of the continuous phase must
be known at the measurement frequency (which is not always the same as that mea-
sured in a conventional viscometer) (Cho and McClements 2007).
Electroacoustic spectroscopy couples ultrasonic and electric fields. Currently,
there are two separate approaches to electroacoustics: the electrokinetic sonic
amplitude (ESA) approach and the colloidal vibration current (CVI) approach. In
the ESA method, a radio frequency signal is applied to an emulsion and the result-
ing acoustic signal generated by the oscillating particles is recorded. Cho and
McClements (2007) demonstrated that the z-potential of oil droplets measured in
situ were consistent with similar measurements based on light scattering (i.e., highly
diluted emulsions). Both techniques were able to monitor the adsorption of pectin
onto the surfaces of b-lactoglobulin-coated droplets as a function of pectin concen-
tration and pH. The major advantage of the EA technique was that it could be car-
ried out in situ without disturbing the equilibrium between adsorbed and nonadsorbed
polyelectrolyte. Nevertheless, the good agreement between the z-potential values
determined by the EA and microelectrophoresis techniques suggested that emulsion
dilution did not cause an appreciable change in polysaccharide partitioning for the
system used in their study. In the CVI approach, the sample is subjected to a longi-
tudinal ultrasonic pulse. The vibrations generated by the planar ultrasonic wave
compressions, in turn, cause the relative motion of particles to the surrounding bulk
phase. This motion perturbs the electrical double layers of the surrounding colloidal
particles and causes an electrical response (CVI). The electrophoretic mobility and
z-potential can be derived from the CVI signal, the ultrasonic properties of the par-
ticles, and the density contrast between the continuous and dispersed phases. Beattie
and Djerdjev (2000) showed that the changes occurring during Ostwald ripening in
emulsions could be followed using electroacoustics. Using an ESA-based method,
Mun et al. (2008) investigated the possibility of using polysaccharide coatings to
improve the freezethaw and freezedry stability of protein-coated lipid droplets. In
that study, emulsions were diluted to a droplet concentration of approximately
0.006 wt.% oil using buffer solution prior to analysis. Diluted emulsions were then
injected into the measurement chamber of a particle electrophoresis instrument and
the z-potential was determined by measuring the direction and velocity that the
droplets moved in the applied electric field. The z-potential measurements were
reported as the average and standard deviation of measurements made on two freshly
prepared samples, with five readings made per sample. Their results show that form-
ing a polysaccharide layer around the protein-coated lipid droplets in an oil-in-
water emulsion cannot prevent destabilization during freezethaw cycling or
freezedrying in the absence of maltodextrin. Nevertheless, a polysaccharide coat-
ing can appreciably decrease the concentration of maltodextrin required to create
emulsions that are stable to freezethawing or freezedrying. This may have impor-
tant consequences for the development of reduced-sugar or reduced-calorie frozen
or powdered emulsion products. Glseren and Corredig (2011) used the CVI
approach to describe oil-in-water emulsions stabilized by sodium caseinate. The
systems were employed as a model to observe the changes in acoustic and electroa-
coustic parameters during different types of flocculation caused by changes in pH or
by the presence of polymers in the continuous phase. The electroacoustic and ultra-
sonic properties of soy oil-in-water emulsions were determined for sodium casein-
atestabilized emulsions under conditions known to cause destabilization. Ultrasonic
attenuation and electrophoretic mobility (z-potential) could clearly follow the
changes occurring in the emulsion droplets, under minimal sample disruption. This
is critical for these systems in a very fragile, metastable state. Destabilization was
generally characterized by a reduction in attenuation, a decrease in electrophoretic
mobility, and an increase in mean particle size. Electroacoustics seemed to be very
sensitive to monitoring structural rearrangements. In detail, the emulsions were
stable to the addition of high-methoxyl pectin (HMP) up to 0.1% HMP. The addi-
tion of free sodium caseinate induced depletion flocculation, causing a decrease in
the attenuation and electrophoretic mobility measured. The presence of HMP lim-
ited depletion interactions. Acidification of the emulsion droplets resulted in a clear
solgel transition, as shown by a steep increase in the particle size and a decrease in
attenuation. Again, destabilization was limited by HMP addition. It was concluded
that ultrasonics and electroacoustics are suitable techniques to understand the details
of the destabilization processes occurring in food emulsions, measured in situ since
different types of destabilizations were monitored and compared with other well-
known techniques such as light scattering measurements.
The fundamental measurements that have been employed in the electroacoustic
and ultrasonic analyses include electroacoustic parameters such as surface charge
3.5 Measurement of Surface Concentration 33
(i.e., z-potential) and electrophoretic mobility of the colloidal particles or ultrasonic

parameters such as velocity and attenuation. All of these parameters have been used
to investigate real-time changes in emulsions and colloids. Other good examples of
applications of these techniques may be found in Corredig et al. (2004b), Dukhin
et al. (2010), and Glseren et al. (2010).
3.5 Measurement of Surface Concentration
Physical properties of oil-in-water emulsions stabilized by milk proteins are deter-

mined largely by the nature of the adsorbed layer at the surface of the dispersed
droplets. The creaming behavior and droplet flocculation are also sensitive to the
concentration of nonadsorbed casein. It was reported that in casein-stabilized emul-
sions, at protein contents around that required for saturation monolayer coverage,
the systems are very stable toward creaming and coalescence (Dickinson 1999).
Thus, measurements of protein surface concentration are very relevant since they
are closely related to the stability of emulsions. Although the interface plays a criti-
cal role, the interfacial protein concentration cannot be studied directly. Kalnin et al.
(2004) presented a new method for the determination of particle surface coverage
by NaCas based on density measurements. In this respect, easy-to-apply techniques
are well suited to study colloids of biological, pharmaceutical, medical, or industrial
interest. In their experiments, emulsions containing 20% fat were stabilized with
14 wt.% NaCas, which leads to a different size distribution using the same homog-
enization pressure. The total amount of NaCas, [NaCas]tot, partitions into the amount
of NaCas at the interface, [NaCas]lip, and the free NaCas, [NaCas]aq, according to
[NaCas]tot = [NaCas]lip + [NaCas]aq

Their results showed that for small amounts of [NaCas]tot, the concentration
seems to limit the fat globule size in the emulsion, whereas for higher concentra-
tions, the homogenization pressure seems to be the limiting factor, since the globule
size distribution does not change significantly at [NaCas]tot > 3 wt.%. This means
that there will be a partition expressed as
K = [NaCas]lip / [NaCas]aq
between the aqueous phase and the interface depending on the surface area of
the interface. They supposed for the further interpretation that the gravitational
force that was apply to reinforce the creaming procedure was too weak to deplete
the fat globules of their [NaCas]lip and only separates the aqueous phase as is
without changing this partition. Therefore, [NaCas]lip was determined by density
measurements of the aqueous phase resulting from the centrifuged emulsion.
Provided that the partition coefficient K is not affected by the centrifugation step,
Kalnin et al.s (2004) new method based on density measurements allows the quan-
titative characterization of NaCas content in the aqueous phase of oil-in-water
emulsions. Once K is known and the mean size of the emulsion droplets has been
measured, the interfacial composition can be determined after measuring the
excess concentration in the aqueous phase.
3.6 Microscopic Analysis
3.6.1 Transmission Electron Microscopy (TEM)
An important part of our understanding of materials is derived from studies of their

behavior under changing conditions of temperature, stress, environment, etc. The
basis of many of these studies is the direct relationship between the microstructure
and the properties of materials, whereby an understanding and characterization of
the former lead to an explanation of the latter. Transmission electron microscopy
has proved to be the most successful instrumental technique for this purpose by
providing structural, morphological, and compositional information from small vol-
umes of thin-foil specimens.
O/W emulsions or W/O creams may be complex multicomponent preparations.
They are combinations of a number of surfactants, polymers, and other additives.
Mixed emulsifiers are frequently used in O/W systems; these are the combinations
of an ionic or nonionic surfactant with fatty amphiphiles, such as fatty alcohols,
fatty acids, or monoglycerides. Berg et al. (2004) developed methods to study W/O
emulsions that make it possible to study the structure of complex multicomponent
mixtures while largely preserving their structure and to predict their stability. The
samples were cryoimmobilized using the jet freezing technique with liquid propane.
In this method, the redistribution of ingredients or phase transitions is avoided in the
products under investigation by using cooling rates of approximately 30,000 K/s.
After cryoimmobilization, the sample was freeze-fractured by using a special frac-
ture holder with specimen sandwiches. It was placed on the precooled specimen
table that allows fracture of the cryoimmobilized sample to form two complemen-
tary fracture areas. Water was sublimated at 173 K and a pressure of 2 107 hPa to
expose surface structures to a depth of approximately 90 nm. An anticontaminator
directly above the sample prevented the contamination of the fresh fracture face by
water recondensation. The internal structures exposed by fracturing and etching
were replicated by means of a thin platinum/carbon replica. First, 2 nm of platinum/
carbon was evaporated onto the surface at an elevation angle of 45. This gave
rise to electron-dense and less electron-dense areas (shadows) according to the
surface topography in the replica that contribute to the contrast in the TEM. The
replica prepared in this manner was then stabilized by additional evaporation of
about 20 nm of carbon onto the surface at an angle of 90. The respective shadow-
casting film thickness was adjusted using an oscillating quartz crystal thickness
monitor. After shadowing, the replica was floated onto organic solvent (ethanol or
acetone) followed by washing in double-distilled water to remove adhering residues.
3.6 Microscopic Analysis 35
Then the samples were placed on a Cu-grid. A complex commercial W/O emulsion
(25 components) with a volume fraction of j = 0.67 together with 12 model emul-
sions with a variable volume fraction j (0.50.86) were studied by TEM. These
studies showed a transition from a particle solution below a critical jc to a close-
packed network above that jc.
With the aim of clarifying the role of the mixed emulsifier in the structure formation
and water binding mode in the case of O/W creams prepared with different surfactants,
Knya et al. (2007) analyzed the swelling behavior of mixed emulsifiers by means of
direct investigation methods such as transmission electron microscopy (TEM) and
X-ray diffraction. A series of ternary surfactant/CSA/water systems were prepared
with a constant ratio of surfactant to fatty alcohol of 1:4 and increasing water contents
25%, 40%, 60%, 70% w/w. The results revealed that the investigated creams had differ-
ent structures from those mentioned in the literature. The surfactant of the mixed emul-
sifier formed micelles, instead of mixed bilayers with the fatty amphiphile.
Cryo-TEM is a widely used technique for the characterization of a variety of
self-assembled nanostructures. In particular, valuable information on the interpar-
ticle structure of cubosomes, hexosomes, micellar cubosomes, and emulsified
microemulsion droplets has been obtained when using this method as a complement
to scattering techniques (SAXS and SANS) and dynamic light scattering (DLS)
(Yaghmur and Glatter 2009). With respect to the significant developments in the
field of electron microscopy regarding the characterization of nanostructured aque-
ous dispersions, recent studies have suggested the use of tilt-angle cryo-TEM and
cryo-FESEM for providing valuable insight into both the interparticle confined
nanostructures and the 3D imaging of dispersed particles. The tilt-angle cryo-TEM
paves the way as a powerful tool for characterizing the internal nanostructure in
various nanostructured dispersions. Controlling the tilt angle allows a differentia-
tion between different nanostructured dispersed particles (cubosomes, micellar
cubosomes, and hexosomes) and also between the two different interiors in observed
cubosome particles. This method could also be useful for distinguishing between
different internal nano-objects within a dispersion. Boyd et al. (2007) introduced the
cryo-FESEM technique for directly investigating the 3D and surface structures of
both nondispersed liquid-crystalline phases and the dispersed cubosome and hexo-
some particles. In that publication, they pioneered the characterization of the 3D
morphology of cubosome and hexosome particles. They demonstrated that the
3D cubosome structure enclosing aqueous water channels agreed well with the pro-
posed mathematical models using a nodal surface representation.
Using microemulsions (ME) as delivery vehicles requires understanding whether
water-insoluble molecules are delivered by an interaction of the ME system with the
dietary mixed micelles (DMM) in the small intestine to give new mixed micelles, or
by alternate paths. To answer this question, Rosner et al. (2010) diluted DMM and
ME systems at various weight ratios. Based on DLS and cryo-TEM, they found a
decrease in the average droplet diameter and an increase in the droplet density per
unit area compared to pure ME systems. Their results showed that DMM and ME
interacted to create MEDMM mixed micelles, providing a potential pathway for
delivering solubilized molecules.
It is well known that the bioavailability of protein and peptide drugs after oral
administration is very low because of their instability in the gastrointestinal tract and
low permeability through the intestinal mucosa. Numerous methods have been
employed to solve this problem, including chemical modification, addition of enzyme
inhibitor or absorption enhancer, conjugation with receptor-recognizable ligand, using
particulate delivery carrier systems, and so on. Nanoparticle is the most commonly
used method because it can protect the drug from the attack of gastric acid or pancre-
atic enzyme and then promote drug absorption by endocytosis or other mechanisms.
Solid lipid nanoparticle (SLN), composed of physiological compatible lipid, has been
used successfully to improve the bioavailability of protein and peptide. Given that
SLN has a solid structure with strong hydrophobicity, gastric acid and protease enzyme
cannot penetrate it and the drug load is protected greatly. Compared with poly(lactic-
co-glycolic acid) (PLGA) nanoparticle, the degradation products of SLN are weaker
acids that have little effect on the stability of protein and peptides. However, due to
their hydrophilic nature, most proteins are poorly encapsulated into the hydrophobic
matrix of SLN, tending to partition in the water phase during the preparation process.
Therefore, the drug-loading capacities are extremely low. Yang et al. (2010) devel-
oped protein-loaded SLN with high entrapment efficiency (EE). For this purpose,
hydrogel was selected as the carrier for protein, due to its hydrophilic nature, to
achieve high entrapment of protein and because hydrogen bonds may be formed
between hydrogel and protein. If hydrogel is implanted into SLN, its compatibility
with protein can be utilized to improve the EE of SLN. The particle sizes and z-poten-
tial were characterized by dynamic light scattering and electrophoretic light scatter-
ing. Transmission electron microscopy (TEM) was employed to investigate the
structure of this gel-core-SLN. The gel-core-SLN was successfully prepared and the
particle size was 305.2 nm, with a z-potential of 17.15 mV. Observations by TEM
confirmed that most solidified hydrogel particles were dispersed in the center of the
gel-core-SLN in the form of a single core, which effectively prevented the diffusion of
proteins to the external water phase during the preparation process.
Traditionally, synthetic surfactants or surface-active polymers, which tend to be
adsorbed onto the interface and thereby reduce the interfacial free energy, were
employed as emulsifiers in the preparation of stable emulsions. With increasing
legal and consumer requirements such as nontoxic, biocompatible, mild to skin,
high ecological acceptability, attractive price-to-performance ratio, and degrees of
freedom in selecting and designing, these classic emulsifiers are becoming more
and more limited. On this background, Pickering emulsions, which are emulsions
stabilized solely by fine solid particles instead of surfactants, have attracted more
and more interest due to their special properties. Pickering emulsions show favor-
able properties in comparison to classic surfactant-stabilized emulsions. The highly
enhanced stabilization against coalescence and Oswald ripening makes it possible
to conserve the droplets under a high concentration of dispersed phase, and they are
even allowed to dry and redisperse. In addition, they are usually insensitive to
changes in chemical parameters such as pH, oil composition, adding electrolytes,
etc. Furthermore, additional stabilization can be achieved when the particles aggre-
gate and form a three-dimensional network in the continuous phase, in which the
3.6 Microscopic Analysis 37
droplets are captured in the array of particles. A wide variety of solid particles,
including organic particles (such as polymer latex and polymer micelle) and inor-
ganic particles (e.g., silica, hydroxides, and clay particles), have been used for the
stabilization of emulsions. Chen et al. (2011) performed rheological studies using
differently modified dispersible colloidal Boehmite alumina nanoparticles.
Differential scanning calorimetry (DSC) results, transmission electron microscopy
(TEM) images, and optical microscopy images of these emulsion systems were
additionally analyzed to reveal the emulsions type and their microstructure.
Pickering emulsion stabilized by moderately hydrophobic particles exhibited an
inhomogeneous structure, relatively large yield stresses, and thixotropic flow behav-
ior, indicating a formation of a three-dimensional network. An emulsion stabilized
by rather hydrophobic particles with a contact angle around 90 was revealed to be
an oil-in-water-in-oil multiple emulsion. The emulsion was homogeneous and
showed thixotropy, indicating the presence of a three-dimensional network.
However, a phase separation slightly occurred in the storage time evaluated, mean-
ing that the stability of this emulsion needs to be improved.
3.6.2 CLSM
Light microscopy is a well-developed and increasingly used technique for studying

the microstructure of food systems in relation to their physical properties and pro-
cessing behavior. Good-quality, high-resolution images of the internal structures of
foods can only be obtained from thin sections of the sample. Procedures that applied
substantial shear and compressive forces may destroy or damage structural ele-
ments, and sectioning is time-consuming and involves chemical processing steps
that may introduce artifacts and make image interpretation difficult. Confocal laser-
scanning microscopy (CLSM) overcomes these problems. In this instrument, image
formation does not depend on transmitting light through the specimen, and, there-
fore, for the first time, bulk specimens can be used in light microscopy. The instru-
ment uses a focused scanning laser to illuminate a subsurface layer of the specimen
in such a way that information from this focal plane passes back through the speci-
men and is projected onto a pinhole (confocal aperture) in front of a detector. Only
a focal plane image is produced, which is an optical slice of the structure at some
preselected depth within the sample. By moving the specimen up and down relative
to the focused laser light, a large number of consecutive optical sections with
improved lateral resolution (compared with conventional light microscopy) can be
obtained with a minimum of sample preparation. CLSM has been used in food sci-
ence since the 1980s. Several reviews discussing the application of this technique in
microstructural studies of food products have been reported in the literature (Heertje
et al. 1987; Blonk and Van Aalst 1993; Marangoni and Hartel 1998). These reviews
have shown the advantages of using CLSM over conventional techniques for study-
ing the relationships among the composition, processing, and final properties of
these products (Herrera and Hartel 2001).
CLSM has also been used to successfully describe emulsions structures. As the
fatty acid esters of propylene glycol are widely used in the manufacturing of food
and cosmetic emulsions, Macierzanka and Szelg (2006) studied microstructural
properties of emulsions prepared with these compounds. Mono-diester products of
the esterifications of propylene glycol with fatty acids were used as stabilizers of
W/O emulsions. The emulsifiers also contained some amounts of unreacted fatty
acids and propylene glycol, as well as zinc carboxylates, the compounds that were
used in order to accelerate the esterification. An interfacial crystallization and the
formation of the continuous oil-phase crystalline network of the acyl propylene
glycols produced very lasting barriers against the sedimentation and coalescence of
water droplets. The relative concentrations of monoacyl propylene glycol/zinc fatty
acid carboxylate, the two main emulsifying components of the emulsifiers used,
influenced the microstructure and, as a consequence, the rheological properties of
the finally obtained W/O emulsions. By increasing the concentration of zinc car-
boxylates, an easier flow of W/O emulsions can be obtained. This is because a dif-
ferent structure of the emulsions is formed for various monoacyl propylene glycol/
zinc carboxylate proportions. This work showed the importance of the microstruc-
ture in stability behavior.
The constituents of foods, or the foodstuffs themselves, are often not regarded as
materials in the conventional sense. However, in reality, they are invariably highly
complex composites that require thorough structural characterization if their physi-
cal behavior is to be fully understood. Only rarely is the actual evolution of the
structure assessed dynamically during such experiments, as the majority of charac-
terization techniques for these soft-solid materials require that the structure is set in
some way prior to examination (i.e., freeze-drying to remove water). Conversely,
the use of CLSM generally allows the materials to be examined in their natural state
(i.e., hydrated) in such a way that structural evolution due to mechanical or thermal
perturbation can be readily followed. Because of this, CLSM is a powerful tool to
study gels. Plucknett et al. (2001) examined mixed biopolymer gels by CLSM, with
the application of dynamic mechanical deformation during visualization. Specifically,
they focused on the large strain deformation and failure behavior of two immiscible
phase-separated composite biopolymer gel systems, namely, gelatin/maltodextrin
and gelatin/agarose. Their results showed that the interfacial fracture energy of the
mixed biopolymer system plays a significant role in determining the failure mode.
For a relatively weak interface, such as for the gelatin/maltodextrin system (when
gelatin is the continuous phase), debonding of the interface occurs at relatively low
strains, resulting in a pseudo-ductile stress/strain response. For the gelatin/agarose
system (again when gelatin is continuous), where the interfacial fracture energy is
an order of magnitude higher, only limited debonding occurs and the material is
nominally linear to failure. Direct evidence for debonding, in both cases, was
obtained by conducting tension and compression tests in situ on the CLSM.
Other gels studied by CLSM were those formulated with whey proteins. Whey
proteins, as important nutritional and functional food ingredients, have been exten-
sively used in various food applications, such as sport beverages, meat replacements,
baked products, salad dressings, ice creams, artificial coffee creams, soups, and vari-
ous dairy products (Dissanayeke and Vasiljevic 2009). These proteins are excellent
3.7 Nuclear Magnetic Resonance (NMR) Techniques 39
foaming and emulsifying agents and easily diffuse to the newly formed wateroil
interface (e.g., during the emulsification), unfold and reorient in ways that greatly
lower interfacial tension, and subsequently form cohesive and viscoelastic films
mainly by disulfide bonds and hydrophobic interactions. Manoi and Rizvi (2008)
showed that the conformational structure and functionalities of whey protein concen-
trate (WPC) could be modified through partial denaturation by means of combined
treatments of highly acid treatment (pH < 3.0) with heat, shear, and supercritical car-
bon dioxide (SC-CO2) injection during a supercritical fluid extrusion process (SCFX)
in the presence of optimum salt concentrations. Compared with the emulsions, the
emulsion gels exhibit some greatly improved characteristics, including improved oxi-
dative stability of lipids and controlled release for bioactives, and thus may have more
potential when applied as the carriers for bioactives. Taking this into consideration,
the heat-set emulsion gels are clearly not suitable as the carrier for heat-labile bioac-
tives, while those obtained by cold-set techniques without heat treatment will be much
more favorable, especially for food formulations containing heat-sensitive ingredi-
ents. Manoi and Rizvi (2009) used a novel supercritical fluid extrusion (SCFX) pro-
cess to texturize whey protein concentrate (WPC) into a product with cold-setting gel
characteristics that was stable over a wide range of temperature. The emulsifying
activity and emulsion stability indices of texturized WPC (tWPC) and its ability to
prevent coalescence of O/W emulsions were evaluated and compared with the com-
mercial WPC80. The cold, gel-like emulsions were prepared at different oil fractions
(j = 0.200.80) by mixing oil with the 20 wt.% tWPC dispersion at 25C and evalu-
ated using a range of rheological techniques. The microscopic structure of cold, gel-
like emulsions was also observed by CLSM. The results revealed that the tWPC
showed excellent emulsifying properties compared to the commercial WPC in slow-
ing down emulsion-breaking mechanisms such as creaming and coalescence. Very
stable with finely dispersed fat droplets and homogeneous O/W gel-like emulsions
could be produced. Liu and Tang (2011) also reported another novel process to pro-
duce cold, gel-like emulsions at various j values in the range 0.20.6 from heat-
treated whey protein dispersions by microfluidization emulsification. The emulsifying
properties of the emulsions formed at low-protein concentrations less than 1% (w/v)
were evaluated. The rheological and microstructural characteristics of the gel-like
emulsions were characterized using steady and dynamic rheological measurements
and a CLSM. CLSM analyses confirmed close relationships between rheological
properties and gel network structures. The formation of the gel-like network structure
was closely related to the high emulsifying efficiency by microfluidization. This kind
of novel gel-like emulsion might exhibit great potential and be applicable in food
formulations, for example, as a kind of carrier for heat-labile and active ingredients.
3.7 Nuclear Magnetic Resonance (NMR) Techniques
There are different types of measurements to characterize emulsion physicochemi-

cal properties that involve NMR measurements: evaluation of relaxation time T2 and
determination of particle size distribution.
Time-domain low-resolution NMR (TD-NMR) relaxation experiments allow the

distribution of the T2 populations in the emulsions to be determined. 1H spinspin
relaxation experiments are usually performed using the Carr-Purcell-Meiboom-Gill
(CPMG) pulse sequence. Typically, a 90 pulse for 2.8 ms followed by a 180 pulse
for 10.0 ms with a recycle delay of 2 s is applied to samples. A true T2, devoid of any
other contribution except that from the sample spin energy exchange, is the time
constant of the exponential that describes the envelope of the echo decay in a CPMG
experiment. A dynamically heterogeneous sample can have its echo decay analyzed
according to a multiple exponential function:
M = M 0 K1e / T21 + K 2 e / T22 + K 3 e / T23 + .. + K n e / T2 n
from which different T2i values, corresponding to dynamically different populations

within the sample, can be calculated. The signal decay represents an envelope of
several T2i, from populations of different motilities that can be described by their
respective T2i values. To do this, data are fitted initially with the sum of discrete
exponential functions, with the best fit given by both the visual adherence of the
fitted function to the experimental curve and the presence of a less structured, more
random residual curve. With this approach, Silva et al. (2010) were able to identify
at least three contributions of different T2i for a rumen or soy protein emulsions cor-
responding to three major distinct populations of protons in respect to their mobil-
ity. The aim of their work was to study the effects of extrusion cooking on rumens
and soy proteins behavior as emulsion stabilizers. The results showed that extru-
sion was able to upgrade functional properties of rumen protein and improve some
characteristics of the emulsions formed with it. The improved emulsion behavior
promoted by rumen extrusion was explained by the molecular reorganization of
protein after processing that considerably increased its hydrophobic surface, increas-
ing its emulsion capacity and improving the emulsion structure. According to the
authors, extrusion can promote the use of rumen, a byproduct waste from the meat
industry, in human nutrition by partially replacing soy protein in food emulsions.
In food science, the description of droplet sizes in quantitative terms is of impor-
tance for quality control and process monitoring as well as fundamental food
research. The size of oil droplets in oilwater (O/W) emulsions such as mayonnaise
and dressings may affect the off-flavor development, flavor release, structure, and
appearance of the products. Particle sizing instruments based on NMR utilize inter-
actions between radio waves and the nuclei of hydrogen atoms to obtain informa-
tion about the microstructure of emulsions (McClements 2007). An emulsion is
placed in a static magnetic field gradient and a series of radio frequency pulses is
applied to it. These pulses cause some of the hydrogen nuclei in the sample to be
excited to higher energy levels, which leads to the generation of a detectable NMR
signal. The amplitude of this signal depends on the movement of the nuclei in the
sample: The farther the nuclei move during the experiment, the greater the reduction
in the amplitude. A measurement of the reduction in signal amplitude with time can
therefore be used to study molecular motion. In a bulk liquid, the distance that a
molecule can move in a certain time is governed by its translational diffusion
3.7 Nuclear Magnetic Resonance (NMR) Techniques 41
coefficient. When a liquid is contained within an emulsion droplet, its diffusion is

restricted because of the presence of the interfacial boundary. If the movement of a
molecule in a droplet is observed over relatively short times, the diffusion is unre-
stricted, but if it is observed over longer times, the diffusion is restricted because the
molecule cannot move farther than the diameter of the droplet. By measuring the
attenuation of the NMR signal at different times, it is possible to identify when
the diffusion becomes restricted and thus estimate the droplet size distribution using
a suitable mathematical model (McClements 2007). Commercial instruments based
on NMR are sensitive to particle sizes between about 200 nm and 100 mm. These
instruments can analyze emulsions with particle concentrations ranging from around
1 to 80 wt.% and can therefore be used to analyze many food emulsions without the
need for sample dilution. The NMR technique is particularly useful at determining
the actual size of the individual droplets in flocculated emulsions (rather than the
floc size), as this technique relies on the molecular movement of water molecules
within droplets and it detects size increases in the droplets themselves and not the
clustering of droplets, thereby differentiating between coalescence and flocculation/
coagulation. NMR-restricted diffusion measurements carried out on the continuous
phase of emulsions can also be used to provide information about the structural
organization of the droplets within flocs. Like ultrasonic spectrometry, NMR is non-
destructive and can be used to analyze emulsions that are concentrated and optically
opaque (McClements 2007). The absence of any sample pretreatment other than
temperature equilibration makes the technique extremely useful to follow changes
in the droplet size of various protein-stabilized O/W model emulsions during pro-
longed storage and temperature cycling. Static light scattering requires extensive
sample preparation to break the covalent bonds that form during storage and is
therefore not a very suitable alternative for these unstable systems. Furthermore, the
interpretation of static light scattering data depends considerably on the correct
choice for the value of the complex refractive index, which may be difficult to obtain
independently for these complex model emulsions. The droplet size results obtained
may also be supplemented with scanning electron microscopy (SEM) imaging
(Kiokias et al. 2004b).
The study of partial coalescence in complex emulsions requires a well-consid-
ered choice of the model system. A typical paper in the field of emulsion science
involves a liquid dispersed phase (e.g., mineral or triacylglycerol oil), which gener-
ally forms a stable emulsion. Moreover, in the case of protein-stabilized emulsions,
preferably neutral and native systems are studied, which tend to be less prone to
partial coalescence, too. In contrast, heated-acidified emulsions prepared with partly
crystalline fat, which can serve as model systems for certain cream cheesetype
products, are far less stable. Kiokias et al. (2004a) studied the stability of heat-
treated and/or -acidified, partly crystalline fatbased, whey proteinstabilized O/W
emulsions against partial coalescence during chilled storage (at 5C) and repeated
temperature cycling (three times between 5C and 25C). They prepared model
O/W emulsions from mixtures of 30% lipid phase consisting of either partly crystal-
line vegetable fat (1:1 mixture of fully hardened coconut oil and fractionated palm
oil, 2.4% solid fat content at 25C, 14% at 20C, 73% at 5C) or liquid vegetable oil
(sunflower oil), 4% powder with 75% protein. Arround 10% of the protein is
denatured. Experiments focused on the evolution of firmness and droplet size (using
pulsed-field gradient NMR and scanning electron microscopy). Besides the effects of
denaturation and/or acidification, the influence of the droplet size of the dispersed
phase on emulsion stability was investigated also. They formulated emulsions in
which the protein content was so high that beforehand destabilization would have not
been expected to occur. Nevertheless, that study showed that such emulsions could
be destabilized through a reduction in protein functionality by heating and/or acidifi-
cation. They studied partial coalescence in two ways: in terms of its microscopic and
its macroscopic effects. Droplet size was chosen as the microscopic property under
investigation. Fat droplet size was known to be related to (partial) coalescence and to
textural characteristics of an emulsion gel, and its evolution can be taken as a mea-
sure for the long-term stability of the emulsion during chilled storage. Droplet size
was studied by means of pulsed-field gradient NMR (pfg-NMR). It was found that
heat treatment or acidification before emulsification led to unstable emulsions during
temperature cycling, whereas heat treatment after acidification resulted in stable
emulsions.
Many water-in-oil (W/O) emulsion-based processed foods (e.g., whipped top-
pings, table spreads, and sauces) are frozen to improve their long-term storage
before thawing for further processing or consumption. In the food industry, freeze/
thaw-induced destabilization is considered unacceptable, as it leads to compromised
consumer acceptability. Both interfacially active and continuous-phase crystals may
play a role in W/O emulsion stability. With the former, colloids collect at the emul-
sion droplet interface, anchor themselves onto the droplet surface, and provide a
physical barrier to coalescence. In foods, so-called Pickering species may originate
by way of surfactant solidification at the interface [e.g., monoacylglycerols (MAGs)]
and/or the migration of previously formed crystals toward the droplet interface.
Crystals lacking any surface activity, but that are present at a sufficiently high con-
centration, will form a plastic network throughout the continuous phase of the emul-
sion, thus encasing the dispersed phase and reducing droplet diffusion and
sedimentation. Oil-in-water (O/W) emulsions are prone to freeze/thaw destabiliza-
tion, given the large volume expansion of the continuous aqueous phase during
freezing. When the oil droplets crystallize prior to the continuous aqueous phase,
the emulsion will destabilize due to the formation of a network of aggregated crys-
talline droplets that coalesce as the dispersed fat phase melts. Ghosh and Rousseau
(2009) investigated the relationship between crystallization and the stability of con-
tinuous- and dispersed-phase W/O emulsions. Two continuous oil phases were
selected based on the order of their crystallization events, either before (coconut oil)
or after (canola oil) the crystallization of the emulsified water phase. As well, two
surfactants were chosen based on their phase behavior: molten [polyglycerol polyri-
cinoleate (PGPR)] or solid [glycerol monostearate (GMS)]. In so doing, continu-
ous-phase crystallization was isolated from that of the dispersed phase as well as
that of the surfactant phase, leading to a clear delineation of the role of interfacial
crystallization versus network crystallization and the role of aqueous phase crystal-
lization on stability. Their hypothesis was that Pickering crystals formed by GMS
3.8 Optical Methods 43
would better stabilize the dispersed aqueous phase than molten PGPR, irrespective
of whether the continuous phase was crystallized or not. Emulsion stability was
assessed with pulsed-field gradient NMR droplet size analysis, sedimentation,
microscopy, and differential scanning calorimetry. This study demonstrated that the
sequence of crystallization events (i.e., dispersed after continuous phase or vice
versa) and the physical state of the surfactant at the oilwater interface strongly
impact the freezethaw stability of water-in-oil emulsions. With the materials and
experimental conditions studied here, freezethaw destabilization of W/O emul-
sions was most apparent with a liquid-state emulsifier and a continuous oil phase
that solidified prior to the dispersed phase (the PGPR-CNO emulsion). Conversely,
emulsions stable to freezethaw cycling were obtained with emulsifier crystallized
at the oilwater interface (the GMSCNO emulsion) or in emulsions where the con-
tinuous phase crystallized after the dispersed aqueous phase (the PGPR-CO and
GMSCO emulsions). These results showed that the relevance of the choice of sur-
factant and composition of the continuous oil phase strongly impact the stability of
W/O emulsions to freezethaw cycling.
3.8 Optical Methods
3.8.1 Dynamic Light Scattering (DLS)
Light scattering (LS) techniques have been applied to many areas of research. There
are several reasons for the rapid development of LS methodology. It is noninvasive;
the sample to be studied does not need to be prepared in any way (except by dilu-
tion), so there are few experimental artifacts. It is relatively easy to use and gives
immediate results; measurements may be made in a few seconds if necessary; and
depending on the sophistication of the analysis, information on the size distribution
is obtained within a short calculation time. The theory of light scattering for a few
well-defined models, such as hard spheres and rods, is well established, and there
are computer models available to convert experimentally obtained data into size
distributions. It is relatively inexpensive to set up and to run. Additionally, results
are very repetitive. For all these reasons, dynamic light scattering (DLS) techniques
have been used to study the stability of suspensions or emulsions and to estimate the
dispersed-phase properties such as particle size and volume fraction. However,
these methods require highly transparent, and in general highly diluted, samples.
Concentrated suspensions, such as milks or food emulsions, may change if they are
diluted into inappropriate media, and great care must be taken to ensure that the
particles under study retain the structures they have in the original foods. In addi-
tion, the high degree of dilution required generally makes it impossible to study
processes that occur only in concentrated media, such as gelation or phase separa-
tion. For this reason, DLS techniques have to be associated with other techniques to
study emulsion stability in food research (Alexander and Dalgleish 2006). In LS
studies, the intensity of the light arriving at the detector at any instant depends on
the interference pattern created by the scattered light from all of the particles in the
scattering volume. This intensity fluctuation provides information on the sizes and
size distributions of the suspended particles, or information on the viscosity of the
sample if the particle sizes are known. Usually, this technique measures some prop-
erty of particles in the analyzed system and assumes that this refers to a sphere, hence
deriving one unique number (the diameter of this sphere) to describe the particle.
This ensures that only one number can be used to describe a 3D particle and make it
unnecessary to describe the particle with three or more numbers, which, although
more accurate, is inconvenient for some purposes, such as, for example, the manage-
ment of products in the food industry. The distribution may be expressed in different
ways depending on the application: the number of particles with a specific diameter;
the surface mean diameter, also called the Sauter mean diameter (d3,2); or the volume
mean diameter, also called the De Brouckere mean diameter (d4,3). In a catalytic reac-
tion, for example, a comparison of particles on the basis of surface area would be very
appropriate because it would be expected that the higher the surface area, the higher
the activity of the catalyst. In this case, results would be expressed in d3,2 values.
According to Relkin and Sourdet (2005), the d4,3 parameter is more sensitive to fat
droplet aggregation than d3,2 (surface weighted diameter). Thus, for evaluation of the
structural stability of a food emulsion against flocculation/coalescence, d4,3 would
be the selected parameter. The advantage of using the parameters d3,2 and d4,3 may be
understood by knowing that in both cases, calculations of the means and distributions
do not require knowledge of the number of particles involved. Particle counting is
normally only carried out when the numbers are very low (in the ppm or ppb regions)
in applications such as contamination, control, and cleanliness. When the distribution
of a system with many small particles and a few big particles is expressed in number
a monomodal graph is obtained. On the contrary if for the same system the distribu-
tion is expressed in volume two peaks, that is, a bimodal distribution is obtained.
Big particles are more notorious when the distribution is expressed in volume.
Relkin and Sourdet (2005) studied four emulsions, stabilized by milk proteins,
before and after application of whipping and freezing procedures. The emulsions
were different in the whey protein/casein ratio and degree of heat denaturation
before mixing with the other ingredients. They found that emulsions with a high
surface protein concentration and a low crystalline fat content showed greater drop-
let aggregation under whipping and freezing. Their results indicated that differences
in sensitivity to fat droplet aggregation were not caused by differences in resistance
to protein displacement from the droplet surface in thawed whipped emulsions, but
by low crystalline fat content prior to whipping and freezing. Data collected in this
study were discussed in terms of the effects of partial replacement of native whey
proteins by casein or heat-denatured whey proteins on partial coalescence of fat
droplets in whipped-frozen emulsions.
For the preparation of milk whipped creams or ice creams, the lipid ingredient
comes mostly from milk fat (in the United States) or vegetable fats (palm oil or
palm kernel oil) in other countries. These lipids consist of a wide diversity of fatty
acids and triacylglycerols (TAG), each characterized by its own melting temperature.
Their chemical and physical properties may be modified by fractionation or

hydrogenation, leading to different characteristic melting profiles and crystalliza-
tion kinetics (Herrera et al. 1999). Bazmi and Relkin (2006) studied the thermal
transitions and physical stability of oil-in-water emulsions containing different
milk fat compositions, arising from anhydrous milk fat alone (AMF) or in mixture
(2:1 mass ratio) with a high melting temperature fraction (HMF) or a low melting
temperature fraction (LMF). The droplet size distribution of these ice cream model
systems was evaluated by dynamic light scattering measurements and fluorescence
microscopic observations. The results indicated differences in fat droplet aggrega-
tion-coalescence under freezethaw procedures, depending on the emulsions fat
composition. It appeared that under quiescent freezing, emulsions containing AMF
LMF were much less resistant to fat droplet aggregation-coalescence than emul-
sions containing AMF or AMFHMF. Their results suggested that the fat droplet
liquidsolid content was very relevant to the emulsions stability.
Semenova et al. (2009) investigated the relationship between the ratio of sodium
caseinate/dextran sulfate used and emulsion stability by combining static and dynamic
light scattering. Various structural and thermodynamic parameters have been deter-
mined for the complex particles formed from sodium caseinate (0.5 wt/vol.%)/dextran
sulfate (0.01, 0.1, or 1.0 wt/vol.%) in aqueous solution at pH = 6.0. The polysaccharide
contents refer, respectively, to three polysaccharide/protein molar ratios (R = 1, 10, and
100) calculated on the basis of the measured values of the weight-averaged molar
masses of sodium caseinate particles and dextran sulfate molecules. The complexes
were prepared by mixing together the two biopolymer components in bulk solution or
bringing them together at the interface in a protein-stabilized foam. The results indi-
cate dissociation of the original sodium caseinate particles in response to associative
interactions with excess amounts of negatively charged polysaccharide. A significant
difference was observed between properties of complexes formed in solution and
those formed at the interface, especially for R = 100. Semenova and colleagues found
a possible correlation between the structures of these complexes and the stability
properties of oil-in-water emulsions containing the same biopolymers, which was
described elsewhere (Jourdain et al. 2008).
Zheng et al. (2011) reported a novel fish oil O/W microemulsion (formed by
nanodroplets) system formulated with food acceptable components, Tween 80,
ethyl oleate, fish oil, and water. The fish oil used was rich in docosahexaenoic acid
(DHA) and eicosapentaenoic acid (EPA), which are known to play a significant role
in nervous system activity, cognitive development, memory-related learning, neuro-
plasticity of nerve membranes, synaptogenesis, and synaptic transmission. The sys-
tems were investigated using dynamic light scattering (DLS), transmission electron
microscopy, and rheological methods. The obtained results indicated that the parti-
cle sizes of spherical droplets in microemulsions depend significantly on the total
oil-phase content, varying from 5 to 198 nm. The rheological measurements showed
that all studied microemulsions followed shear thinning behavior. Release experi-
ments confirmed that the microemulsion system is potentially useful as a release/
delivery system of fish oil.
3.8.2 Diffusing Wave Spectroscopy (DWS)
In recent years, great strides have been made in our understanding of colloidal inter-
actions in food emulsions. Traditional experimental techniques such as light scatter-
ing, rheology, microscopy, and turbidity have been crucial in determining the forces
at play in complex food structures. In the past 20 years, a new light scattering tech-
nique called diffusing wave spectroscopy (DWS) has been increasingly employed in
the study of highly turbid food media. Its main advantage is the ability to extract
structural and dynamic information between food components in a noninvasive way.
DWS is now gaining pace in its application in the food industry. Much of this suc-
cess can be attributed to the relatively simple theoretical model and rather inexpen-
sive and straightforward experimental setup. DWS is very similar to traditional
dynamic light scattering (DLS) since both techniques follow the temporal fluctua-
tions of intensity of a speckle of scattered light. In both cases, this fluctuation reflects
the dynamics of the scattering particles. This fluctuation has a characteristic time
scale inversely proportional to the particle diffusion coefficient; for this reason, it
can provide information about colloidal particles in the range of tens of nanometers
to a few microns. However, conventional light scattering requires the sample to be
highly diluted (to be in what is called the single scattering regime), and this can
severely restrict its use in industrially relevant systems. In contrast to DLS, DWS
must operate in a highly turbid medium (or multiple scattering regime) because it
treats the photon path through the sample as a diffusive process. Similar to DLS,
DWS is able to provide information about the local dynamics of particle dispersions
without restrictions on particle concentration and turbidity. This is highly desirable
in systems such as foods and soft materials, where most of the colloidal dispersions
(milk, salad dressing, acidified dairy drinks, etc.) are usually quite opaque. There
are two different applications of DWS: the backscattering mode and the forward-
scattering mode. The main difference between them arises in the placement of the
collector system (lens and fiber optics). In the first case, the collector and detector
are placed on the same side as the incident laser light. In the latter case, the collector
and detector are placed on the opposite side of the incident light; that is, the scat-
tered light must travel the whole thickness of the sample, L, before it is detected and
analyzed. There is a formal similarity between the two methods, but there are sig-
nificant differences in the interpretation of the results. This topic was extensively
reviewed by Corredig and Alexander (2008). The interest in using the method is in
studying changes in the behavior of a suspension when it is destabilized by one
means or another (Alexander and Dalgleish 2006; Liu et al. 2007b).
Structure is an important parameter for many food products. To control the final
structure of a food product, one has to understand not only the structuring properties
of different food components but also the influence of the different processing steps.
Gelation has been described as the growth of clusters of particles leading to a space-
filling network. This process succeeds only if the particles are present above a criti-
cal concentration, the attractive interparticle interactions are of sufficient strength,
and the applied stress is sufficiently low. In general, the dynamics of colloidal
systems that are driven away from equilibrium by the application of shear flow are
determined by the interplay of the intrinsic relaxation of the system with shear flow.
The particle motion is often studied using light scattering techniques such as
dynamic light scattering. An extension of this technique for concentrated, turbid
samples is diffusing wave spectroscopy (DWS). DWS has been successfully applied
to the study of interactions of dairy proteins as well as the kinetics of aggregation in
gelling systems under different destabilizing conditions. Ruis et al. (2008) studied
the influence of shear flow on the acid-induced aggregation of a food-based system
that consists of an oil-in-water (O/W) emulsion stabilized by sodium caseinate.
Slow acidification down to pH 4.6, equal to the isoelectric point of sodium casein-
ate, causes the aggregation of emulsion droplets. Understanding the aggregation
behavior and the effect of shear as a function of the pH of such emulsions is relevant
to the food industry, especially the dairy industry. Their results showed that the
emulsion droplets in the food-related emulsion were uniformly dispersed at neutral
pH. Upon acidification down to a pH of 5.2, the emulsion showed Newtonian behav-
ior with constant viscosity over the whole pH range. At pH 5.17, independent of the
applied shear rate during acidification, the viscosity suddenly increased. From this
point on, the emulsion showed shear-thinning behavior. In the DWS method, an
important parameter is the photon-transport mean free path, l*. The value of l* can,
in the absence of absorption, be determined using the average intensity of the trans-
mitted light of a sample combined with the transmission, T, of a reference sample
with known l* by
T=
I
=
(
5l * /3L)
I0 (
1 + 4l * /3L )
In this equation, the transmission is equal to the ratio of the incoming and trans-
mitted energy flux, which is related to the laser (I0) and the transmitted (I) average
light intensities, and L is the path length. For completely noninteracting scatterers,
which are spatially completely uncorrelated, the value of l* depends on the wave-
length of the laser light and on the size, concentration, and optical contrast of the
scatterers with the dispersion medium. The photon-transport mean free path (l*)
was not influenced by the applied shear rate and did not change down to pH 5.2.
Close to this pH, l* increased, and at a lower pH (5.05), l* started to fluctuate.
Assuming that the convective motion and the Brownian motion are independent of
each other, the mean-square displacement as a result of Brownian motion was deter-
mined. From this, the solgel point and the radius of the aggregates at this point as
a function of the shear rate were determined. The results indicated that the radius of
the aggregates at the solgel transition decreased with increasing shear rate and sug-
gested that shear will result in a more open structure of the network formed by the
aggregates. This study shows that DWS is a promising technique for the study of
food systems, even under shear, that are normally very hard to probe by light scat-
tering techniques because of their turbidity.
Polysaccharides are often added to food systems as they impart unique
and desirable textures and micro-structural properties. Pectin, in particular, is
commonly used as a food ingredient because of its unique properties such as

water-holding capacity, viscosity improvement, and gelling properties (Bonnet
et al. 2005; Liu et al. 2007c). Pectin is often added to milk as it provides stabil-
ity to acidified milk products against whey separation. Pectin is an anionic poly-
saccharide found in plant cell walls and is most widely extracted from citrus
peel and apple pomace. It consists of water-soluble, relatively elongated poly-
meric molecules with carboxyl groups. Its structure is formed by rhamnogalac-
turonans as well as homopolymeric poly-a-(1 4)-D-galacturonic acid with
a-(1 2)-L-rhamnosyl-(1 4)-D-galacturonosyl sections containing branch
points with mostly neutral side chains. Some of the carboxyl acid groups in
galacturonans can be methyl-esterified and the amount that is substituted is
referred to as the degree of esterification Pectins with a degree of esterification
of 43% or more are classified as high-methoxyl pectins (HMP), whereas those
with a lower number of methyl esters are called low-methoxyl pectins (LMP).
Acero Lopez et al. (2009) investigated the effect of the addition of different
HMP concentrations on the stability of skim milk and on the renneting kinetics
of casein micelles at pH 6.7. Noninvasive techniques such as diffusing wave
spectroscopy (DWS) and ultrasonic spectroscopy (US) were used to closely
observe the structure formation during renneting in the presence of HMP. These
two techniques allow for in situ measurements of solgel transitions without the
need for dilution and provide information about particle size and mobility, as
well as interparticle interactions that might occur under destabilizing condi-
tions. Their results showed that at low-HMP concentrations, the casein micelles
aggregation behavior was similar to that of skim milk, although changes could
be noted in the microstructure of the renneted gels. At HMP concentrations
between 0.1% and 0.15%, phase separation kinetics were slower than the ren-
net-induced aggregation, and different microstructures formed caused by differ-
ent dynamics of interactions between casein micelles present in HMP-depleted
flocs. Higher amounts of HMP failed to create a continuous gel, as phase sepa-
ration occurred at a faster rate than rennet aggregation. Acero Lopez et al.s
(2009) work highlights the importance of noninvasive techniques in the study of
concentration-dependent phase-separating and -aggregating systems, as only
with observations in situ is it possible to determine new ways to control the
structuring of proteinpolysaccharide mixed systems. These authors also proved
that DWS and the attenuation of sound measured by US were in good agree-
ment, and both identified well the structural changes occurring in the emulsions
under destabilizing conditions (Liu et al. 2008; Gaygadzhiev et al. 2009; Acero
Lopez et al. 2010; Chappellaz et al. 2010).
3.8.3 Turbiscan
Emulsions have been studied by numerous techniques, such as particle sizing,

microscopy, and rheology, among others, to characterize their physical properties.
Most of these techniques involve some form of dilution. This dilution disrupts some
Fig. 3.2 Scheme of the TMA 2000 analyzer
structures such as flocs that contribute to destabilization. The ability to study the
stability of food emulsions in their undiluted forms may reveal subtle nuances about
their stability. A relatively recently developed technique, the Turbiscan method,
allows us to scan the turbidity profile of an emulsion along the height of a glass tube
filled with the emulsion, following the fate of the turbidity profile over time. These
profiles constitute the macroscopic fingerprint of the emulsion sample at a given
time. The analysis of the turbidity profiles leads to quantitative data on the stability
of the studied emulsions and allows us to make objective comparisons among dif-
ferent emulsions (Chauvierre et al. 2004).
Figure 3.2 shows a scheme of the vertical scan analyzer Turbiscan MA 2000.
This equipment allows the optical characterization of any type of dispersion (Pan
et al. 2002; Mengual et al. 1999). The reading head is composed of a pulsed near-IR
light source (l = 850 nm) and two synchronous detectors. The transmission detector
receives the light, which goes through the sample (0), while the backscattering
detector receives the light back-scattered by the sample (135). The Turbiscan head
acquired T and BS data every 40 mm along the vertical length of the cell. Therefore,
the scan of a 60-mm height sample provides patterns, including 1,500 points of
measurement, in less than 20 s. Thus, by repeating the scan of a sample at different
time (t) intervals, the stability or instability of dispersions can be study in detail. The
samples are placed in a flat-bottomed cylindrical glass measurement cell and
scanned from the bottom to the top. The backscattering (BS) and transmission (T)
profiles as a function of the sample height (total height = 60 mm) are obtained in
quiescent conditions usually at ambient temperature. The profiles allow the calcula-
tion of creaming, sedimentation, or phase separation rates, as well as flocculation,
and the mechanism making the dispersion unstable can be deduced from the trans-
mission or backscattering data (Chauvierre et al. 2004).
30
20
10
BS(%)
0
-10
-20
-30
0 20 40 60 80
Tube Length (mm)
Fig. 3.3 Changes in back scattering (BS) expressed in reference mode profiles (subtracting the
profile at t = 0), as a function of the tube length with storage time (the emulsion was stored for
1 week; arrow denotes time) in quiescent conditions. It was formulated with 10 wt.% sunflower oil
as fat phase, 20 wt.% sucrose added to the aqueous phase, and a concentration of sodium caseinate
(NaCas) of 0.5 wt.%. Tube length = 65 mm
The curves obtained by subtracting the BS profile at t = 0 from the profile at t

(DBS = BStBS0) display a typical shape that allows a better quantification of creaming,
flocculation, and other destabilization processes. Figure 3.3 gives an example of a typi-
cal profile of an emulsion that destabilized mainly by creaming of small particles in
direct and reference mode. Creaming was detected using the Turbiscan as it induced a
variation of the concentration between the top and bottom of the cell. The droplets
moved upward because they had a lower density than the surrounding liquid. When
creaming takes place in an emulsion, the DBS curves show a peak at heights between
0 and 20 mm. The variation of the peak width, at a fixed height, during the studied time,
can be related to the kinetics of migration of small particles (Mengual et al. 1999). The
creaming destabilization kinetics may be evaluated by measuring the peak thickness at
50% of the height at different times (bottom zone). The slope of the linear part of a plot
of peak thickness vs. t gives an indication of the migration rate. Figure 3.4 gives an
example of peak thickness vs. t for the emulsion in Fig. 3.3.
For emulsions that destabilize mainly by flocculation, BS mean values (BSav)
change with the increase in particle size. Flocculation is followed by measuring the
BSav as a function of storage time in the middle zone of the tube. As was theoreti-
cally demonstrated by Mengual et al. (1999), the BS intensity decreased as the par-
ticle size increased [when particle size is higher than the wavelength (l) of the
incident light]. It should be mentioned that if the particle size is lower than l of the
incident light, BS increases with particle size. This phenomenon was used by sev-
eral authors to determine flocculation kinetics (Chauvierre et al. 2004; Palazolo
et al. 2005). The optimum zone is the one not affected by creaming (bottom and top
of the tube), that is, the 2050-mm zone. Figure 3.5 shows a typical profile of an
emulsion that destabilized mainly by flocculation in direct and reference mode. The
actual profile of a flocculated emulsion shows a significant change in BSav in the
middle of the tube and its shape at the top of the tube is different from that of migra-
tion of individual particles, which is a consequence of the migration of flocs formed
Fig. 3.4 Peak thickness 20

measured at 50% of the 18 R2 = 0.999
Peak Thickness (mm)

height at different times in 16
the bottom zone of the tube, 14
for emulsion in Fig. 3.3 12
stored in quiescent conditions 10
at 22.5C, monitored over
8
30 min. Tube length = 65 mm
6
4
2
0
0 0.1 0.2 0.3 0.4 0.5
Time (h)
40
30
Back Scattering (%)
20
10
0
10 0 10 20 30 40 50 60 70 80
20
30
40
Tube length (mm)
Fig. 3.5 Changes in back scattering (BS) in reference mode profiles, as a function of the tube
length with storage time (samples were stored for 1 week; arrow denotes time) in quiescent condi-
tions for the emulsion formulated with 10 wt.% sunflower oil as the fat phase, no sugar added to
the aqueous phase, and a concentration of sodium caseinate (NaCas) of 5.0 wt.%. Tube
length = 65 mm
during flocculation. In the case of flocculation, the BS profile in reference mode

does not show a peak at the bottom of the tube (020 mm), which indicates that
there is no detectable migration of small particles.
Several food systems have been studied using a Turbiscan. lvarez Cerimedo
et al. (2010) described the stability behavior of emulsions formulated with high
concentrations of sodium caseinate and trehalose. The emulsion were formulated
with 10 wt.% oil (concentrated fish oil, CFO; sunflower oil, SFO; or olive oil, OO),
sodium caseinate concentrations varying from 0.5 to 5 wt.%, giving oil-to-protein
ratios of 202, and 0, 20, 30, or 40 wt.% aqueous trehalose solution. Those systems
showed a more complicated behavior than that reported in literature. The oil-to-
protein ratio that gave stability changed with processing conditions and formulation
of the aqueous phase. Many studies in the literature evaluated emulsions by visual
observation. By using this approach, all our emulsions would be considered stable
since no phase separation occurred after a week in quiescent conditions at 22.5C.
This is in agreement with the fact that the transmission detector received no light
during the time emulsions were analyzed. However, the BS detector was able to
quantify creaming or flocculation in these systems. Concentrations below 0.5 wt.%

NaCas seemed to be below the ones required for saturation monolayer coverage
since the creaming rate is greater for 0.5 wt.% than for 1 wt.% NaCas. Systems
stabilized by these oil-to-protein ratios most likely destabilized by time-dependent
bridging flocculation. Further addition of protein led to high instability. The
24 wt.% NaCas range was the worst situation. At these protein contents, the cream-
ing stability of the system was greatly reduced probably due to depletion floccula-
tion of protein-coated droplets by unabsorbed submicellar caseinate. Although it
was reported that at protein contents around that required for saturation monolayer
coverage, the system was very stable toward creaming and coalescence (Dickinson
1999), there was no such oil-to-protein ratio in our systems between bridging floc-
culation and depletion flocculation destabilization processes. At even higher protein
contents (5 wt.% NaCas concentration), emulsions were very stable especially when
the aqueous phase contained trehalose. In these conditions, the systems remained in
the liquid state for at least a week, fully turbid; that is, no changes were noticed by
visual analysis, and no creaming or flocculation was detected by the Turbiscan.
Although there was no gel formation when emulsions were kept at 22.5C for a
week, they were very stable. Confocal images obtained after 24 h at 22.5C were in
agreement with Turbiscan data. Turbiscan allowed quantification of creaming and
coalescence kinetics and more objective comparisons.
Cabezas et al. (2011) evaluated the emulsifying properties of different modified
sunflower lecithins in oil-in-water (O/W) emulsions. They studied five modified
sunflower lecithins, which were obtained by deoiling (deoiled lecithin), fraction-
ation with absolute ethanol (PC- and PI-enriched fractions), and enzymatic hydro-
lysis with phospholipase A2 from pancreatic porcine and microbial sources
(hydrolyzed lecithins). Modified lecithins were applied as an emulsifying agent in
O/W emulsions (30:70 wt/wt), ranging from 0.1% to 2.0% (wt/wt), and the stability
of different emulsions was evaluated through the evolution of backscattering pro-
files (%BS), particle size distribution, and mean particle diameters (D4,3, D3,2). The
PC-enriched fraction and both hydrolyzed lecithins presented the best emulsifying
properties against the main destabilization processes (creaming and coalescence)
for the analyzed emulsions. These modified lecithins represent a good alternative
for the production of new bioactive agents.
Juliano et al. (2011) enhanced the creaming of milk fat globules in milk emul-
sions by the application of ultrasound and detected instability using the Turbiscan
method. Coarse and fine recombined emulsions and raw milk were subjected to
ultrasound for 5 min at 35C. Scans were performed over a 10-min period after
removing the samples from the ultrasonic setup. For each model system, an unsoni-
cated control was treated exactly in the same way as the sonicated samples. They
were filled into the Turbiscan tube, put into the water bath, and after 5 min placed
into the Turbiscan; measurements were performed for an additional 10 min. Another
way to quantify creaming destabilization by Turbiscan is the one used by Juliano
et al. (2011). Due to the increased backscattered light at the layers with a higher
number of dispersed particles, it is possible to identify high-cream-concentration
regions. Since the Turbiscan can measure highly concentrated solutions (up to
95% v/v), no significant nonlinear effects are introduced due to high particle
concentrations. The backscattering is directly proportional to the square root of
the concentration only when the particle size is unchanged. To compare the different
creaming extents, the ratio of the area of the peak corresponding to the separated
cream phase was calculated with respect to the total backscattering measured in the
sample. The cream phase was defined as the curve area, starting where the backscat-
tering value is greater than 1.5% in comparison to the baseline value representing
the bulk liquid. The 1.5% value represents the measurement error of backscattering
determined for the Turbiscan equipment. For comparison between different mea-
surements, the area under the cream phase curve was related to the area under the
whole curve. The creaming extent (CE) can be expressed as

L
BS( z )cream phase dz
CE = 0

L
BS( z ) dz
0
where z represents the axial position in the tube (origin at the bottom), L the length
corresponding to the free surface, and BS(z) the backscattering at position z. This
approach assumes that the backscattering, which is related to the number and size
of particles, is shifted when a cream phase is formed. When the particle size of the
dispersions at the time of measurement is unchanged, the calculated creaming extent
provides an indication of the concentration of fat in the creamed phase. However,
when the particle size of the dispersions at the time of measurement is increased,
then the backscattering may be either an overestimate or an underestimate of con-
centration. This is because there is a change in the behavior of the backscattering
around the wavelength of the incident light, where backscattering increases with
particle size up to ~1 mm and then decreases with further increases in particle size.
The calculation of a recovery ratio (RR) computed with the following equation
allows an assessment of the validity of the calculated creaming extent.

L
BS( z )sample dz
RR = 0

L
BS( z )control dz
0
BS(z)sample stands for the backscattering of the sample and BS(z)control for the cor-
responding nonultrasonic treated control at the same time. If this value is close to
unity, it means that the overall backscattering of the sample and the control match.
Assuming no change in particle sizes, a recovery ratio of 1 can be attributed to the
fact that all particles may have moved to another position in the tube but were not
flocculated or coalesced at the time the measurement was taken. However, if there
is a change in particle size, the recovery ratio is the sum of the relative contributions
of the differently sized particles to the backscattering. If there is flocculation/coales-
cence that results in larger particles (i.e., greater 1 mm), backscattering will be
reduced, and the recovery ratio can be less than 100%. If the recovery ratio is greater
than 100%, this can be because of particle size increase due to flocculation/coales-
cence under conditions where the particle size of the floccules/coalesced fat is equal
to or less than 1 mm. In Juliano et al.s systems, creaming, as calculated from

Turbiscan measurements, was more evident in the coarse recombined emulsion and
raw milk compared to that of the recombined fine emulsion. Micrographs confirmed
that there were flocculation and coalescence in the creamed layer of emulsion.
Coalescence was confirmed by particle size measurement. Particle flocculation and
clustering were detected in both the coarse emulsion and raw milk upon sonication,
the extent of which was dependent on the conditions of sonication. Turbiscan was a
successful tool to follow destabilization processes in milk emulsion systems under
high-frequency ultrasonic waves.
There is also a newly delivered Turbiscan online that allows monitoring and
characterization of processes involving emulsions, suspensions, or foams, such as
emulsification (salad dressings, mayonnaise), crystallization (sugar), or bulking
(foams). Buron et al. (2004) reported that the average droplet size calculated from
online measurements with Turbiscan were in agreement with light scattering mea-
surements in the same systems. Measurements were accurate in a wide range of
particle volume fraction (060%) and particle size (0.011,000 mm). Pizzino et al.
(2009) performed an online light backscattering tracking of the transitional phase
inversion of emulsions. In their study, a surfactant/oil/water system (Brij 30/decane/
brine) was stirred while continuously changing the temperature in order to induce
the swap of the emulsion morphology from O/W to W/O, or vice versa. The transi-
tional phase inversion was detected by monitoring both the electrical conductivity
and the light backscattering with Turbiscan (online) equipment. They found that
light backscattering was a complementary technique to conductivity measurement
to track the emulsion phase inversion. Provided that the experimental conditions are
optimized, it may be considered an easy-to-use, noninvasive, and accurate method
of detection with some advantages over the classical conductivity measurement:
(1) It does not require any addition of electrolyte in the aqueous phase; (2) it pro-
vides some information on the drop size; and (3) it works with both O/W and W/O
morphologies, whereas conductimetry is blind for the latter case.
Many industrial processes involve shear-induced dispersion or agglomeration of
suspensions, especially for the production of pigments, stains, or varnish as well as
for the development of cosmetics, pharmaceutical products, or food products. In
such processes involving concentrated micron and submicron particle suspensions,
the control of flocculation processes and suspension microstructural changes
remains a problem of relevance. Now, industrial inspection is usually performed
either after dilution or using intrusive techniques that may induce a denaturing of
the medium. Bordes et al. (2003) developed a nonintrusive and nondenaturing opti-
cal method based on multiple light scattering phenomena. This device was used to
describe in situ the physical properties of a flowing concentrated latex aqueous sus-
pension. An experimental setup, comprising the optical sensor, the Turbiscan read-
ing head, directly mounted inside a Couette flow system, was specifically designed
to study both shear- and time-dependent microstructural changes to a stabilized
latex suspension. Reversible depletion flocculation induced by the addition of a
polymer aqueous solution was also investigated. Scattering parameters of the sus-
pension are derived from measurements of the backscattering level within the
References 55
framework of a mean field scattering model based on the photon diffusion approxi-
mation. Shear flow was shown to promote hydrodynamic melting of crystallites in
a stabilized concentrated latex suspension. The depletion flocculation kinetic of
24% latexCMC (sodium carboxymethylcellulose) suspension was further investi-
gated in Couette flow, and the time dependence of the transport mean path was
determined during cluster growth. As a conclusion, the rheo-optical system is well
suited to study flocculation processes and monitor aggregation kinetics in concen-
trated suspensions.
The Turbiscan method was also used to study nanosystems. Lemarchand et al.
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copolymers varying both the molecular weight and the proportion by weight of
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technique derived from emulsionsolvent evaporation, during which emulsion sta-
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understanding of the ability of the copolymers to migrate to the solventwater inter-
face, a means of following the procedure of nanoparticle preparation and of deter-
mining the factor of coalescence (Fc) of the emulsion droplets during the evaporation
step. This parameter was defined as follows:
xv
(dNP | dd )
3
Fc =
m
where r is the density of the PCL-DEX nanoparticles, m the copolymer weight,
v the volume of ethyl acetate, dNP the mean diameter of the nanoparticles after sol-
vent evaporation, and dd the mean diameter of the emulsion droplets. Because of
their strongly amphiphilic properties, the PCL-DEX copolymers were able to stabi-
lize O/W emulsions without the need for additional surfactants. Nanoparticles with
a controlled mean diameter ranging from 100 to 250 nm were successfully pre-
pared. Zeta-potential measurements confirmed the presence of a DEX coating.
Taking into account the actual relevance of nanoscience, this technique has great
potential for studying a wide variety of systems.
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Index
L N
Lipid emulsions, 1, 32 Nano and microsizes, 9
M P
Methods to study stability, Preparation, 3, 8, 11, 15, 18, 21, 22, 24, 29,
1555 34, 36, 37, 41, 44, 55

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M.L. Herrera, Analytical Techniques for Studying the Physical Properties 1

Fig. 1.1 Confocal laser

undesirable (McClements 2005b). Figure 1.1 shows an example of a confocal laser

Fig. 1.2 Confocal laser

2.1 Methods of Formation

Emulsions with microdroplets, sometimes called conventional emulsions, and

M.L. Herrera, Analytical Techniques for Studying the Physical Properties 7

2.1.2 Conventional Emulsions

Conventional emulsions, that is, emulsions with microdroplets, are thermodynami-

Fig. 2.1 Scheme of conventional emulsions preparation procedure

and Li 2010). Figure 2.1 illustrates a preparation procedure. This is a common

2.2 Physical Chemical Properties

The physical properties of nanoemulsions can be characterized by the combination

by rheometer, conductivity meter, and pendant drop tensiometer, respectively

2.2.2 Conventional Emulsions

Conventional emulsions may also be characterized by the techniques mentioned above.

2.3 Structuring Food Emulsions

The structure of the different pseudoternary phases can be investigated by small-

The efficient development and production of high-quality emulsion-based products

3.1 Visual Observation

Emulsion systems have minimal thermodynamic stability and tend to phase-separate.

M.L. Herrera, Analytical Techniques for Studying the Physical Properties 15

The creaming index is an indirect indication of the extent of droplet aggregation

water-soluble polymer with a few hydrophobic groups in the hydrophilic macromo-

sunflower oil emulsions in the presence of polysaccharide mixtures, using chitosan

the homogenization process (rotor/stator device and high-pressure homogenizer)

3.2 Rheological Methods

3.2.1 Small-Deformation Rheology

therefore tend to result in a greater reduction in apparent viscosity as more of the

Viscosity increase is a common strategy to enhance emulsion stability. This goal

emulsions by using a combination of surfactant and polyelectrolytes (Stamkulov

3.3 Ultrasound Profiling

In a pioneering work, Dickinson and Golding (1997b) followed the stability of

branched regions containing arabinose, galactose, and xylose. Changes in charge

on the volume fraction or minimal shear disturbance. In addition, most of these

3.4 Electroacoustic Spectroscopy: z-Potential

(i.e., z-potential) and electrophoretic mobility of the colloidal particles or ultrasonic

3.5 Measurement of Surface Concentration

Physical properties of oil-in-water emulsions stabilized by milk proteins are deter-

[NaCas]tot = [NaCas]lip + [NaCas]aq

3.6 Microscopic Analysis

3.6.1 Transmission Electron Microscopy (TEM)

An important part of our understanding of materials is derived from studies of their

Light microscopy is a well-developed and increasingly used technique for studying

3.7 Nuclear Magnetic Resonance (NMR) Techniques

There are different types of measurements to characterize emulsion physicochemi-

Time-domain low-resolution NMR (TD-NMR) relaxation experiments allow the

M = M 0 K1e / T21 + K 2 e / T22 + K 3 e / T23 + .. + K n e / T2 n

from which different T2i values, corresponding to dynamically different populations

coefficient. When a liquid is contained within an emulsion droplet, its diffusion is

3.8 Optical Methods

3.8.1 Dynamic Light Scattering (DLS)

Their chemical and physical properties may be modified by fractionation or

3.8.2 Diffusing Wave Spectroscopy (DWS)

commonly used as a food ingredient because of its unique properties such as

Emulsions have been studied by numerous techniques, such as particle sizing,